APOE, ACT and CHRNA7 genes in the conversion from amnestic mild cognitive impairment to Alzheimer's disease

Neurobiology of Aging 30 (2009) 1254–1264

APOE, ACT and CHRNA7 genes in the conversion from amnestic mild cognitive impairment to Alzheimer’s disease A. Barabash a , A. Marcos b , I. Anc´ın a , B. V´azquez-Alvarez a , C. de Ugarte a , P. Gil c , C. Fern´andez d , M. Encinas a , J.J. L´opez-Ibor e , J.A. Cabranes e,∗ a

Laboratory of Psychoneuroendocrinology & Genetics, Hospital Cl´ınico San Carlos, 28040 Madrid, Spain b Department of Neurology, Hospital Cl´ınico San Carlos, 28040 Madrid, Spain c Department of Geriatric, Hospital Cl´ınico San Carlos, 28040 Madrid, Spain d Department of Epidemiology, Hospital Cl´ınico San Carlos, 28040 Madrid, Spain e Department of Psychiatry, Hospital Cl´ınico San Carlos, 28040 Madrid, Spain Received 7 May 2007; received in revised form 5 October 2007; accepted 4 November 2007 Available online 19 December 2007

Abstract We have investigated whether the −86 C/T promoter polymorphism in CHRNA7 gene, the signal peptide polymorphism of the ␣1antichymotripsin (ACT) gene or the APOE genotype are associated with an increased risk of mild cognitive impairment (MCI) or affect the risk of evolution to Alzheimer’s disease (AD). We have followed up 89 patients with initial diagnoses of amnestic MCI for 49 months. Patients were separated into three groups: 27 subjects who remained with MCI, 40 that converted to AD before 20 months and 22 that converted to AD after. To assess the risk associated to each genotype a control group (n = 90) without cognitive impairment was included. APOE4 allele was associated with an increased risk of MCI (OR: 6.04, 95% CI: 2.76–3.23; p < 0.001) but did not have an effect on the probability of evolving AD. ACT or CHRNA7 genotypes were not associated with MCI but both appear to modify the risk of progression to dementia in opposing manners: ACT polymorphism increasing the risk to evolve to AD before 20 months (HR = 2.03; 95% CI: 1–4.6; p = 0.06) and CHRNA7 polymorphism protecting from evolution to dementia. Cox regression model demonstrated that ACT genotype confers a higher risk of rapid evolution to dementia than age or years of schooling. We conclude that APOE is a risk gene for amnestic MCI and that ACT and CHRNA7 may act in these patients as modifier genes for the time of progression to AD. © 2007 Elsevier Inc. All rights reserved. Keywords: Mild cognitive impairment; Alzheimer’s disease; Genetics; APOE; CHRNA7; ACT; Prediction

1. Introduction Dementia is one of the most devastating diseases in developed countries. The number of people suffering from this affliction will increase markedly during the next few decades and it is expected that within the next 50 years there will be approximately 16.2 million people affected by this disorder in the whole European region (Wancata et al., 2003). This may result in an enormous financial and emotional burden ∗ Corresponding author at: Instituto de Psiquiatr´ıa y Salud Mental, Hospital Cl´ınico San Carlos, C/Mart´ın Lagos s/n., 28040 Madrid, Spain. Tel.: +34 91 330 3808; fax: +34 91 330 3574. E-mail address: [email protected] (J.A. Cabranes).

0197-4580/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2007.11.003

for society and particularly for caregivers. There are only palliative treatments available that can benefit some patients for a period of time when the disease is established (Evans et al., 2004). In contrast, we have evidence that pharmacologic intervention at an early stage may delay or prevent progression to dementia (Birks, 2006). Thus, current research in this matter is focused in identifying this initial period of illness. Mild cognitive impairment (MCI) is a clinical diagnostic entity that may represent this early stage. It refers to individuals with cognitive deficits but who do not fulfil a diagnosis of dementia (Petersen et al., 2001). A considerable number of subjects with MCI develop dementia while a relative few do not; that has spurred major therapeutic interest in establishing preclinical prognostic markers for the progression from

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MCI to dementia (Larrieu et al., 2002; Palmer et al., 2002). Among the implicated factors described, some candidate genes involved in the pathogenesis of the disease, alone or in conjunction, may affect the risk of developing dementia and can be considered as susceptibility markers in early stages of memory impairment. Given that the ␧4 allele on the apolipoprotein E (APOE) gene has been well established as a risk factor for AD, most genetic studies among patients with MCI have focused in finding out its involvement in this disease. Some authors have reported that this allele is associated with different degrees of memory impairment in elderly subjects and that it is more frequent among individuals with memory decline over time or among patients who develop dementia when compared with those who do not (Coria et al., 1995; Dik et al., 2000; Rosich-Estrago et al., 2004). A few prospective longitudinal studies of patients with MCI followed over more than a 2-year period, found that the four-allele status was a reliable predictor of clinical progression to dementia (Petersen et al., 1995; Tierney et al., 1996). However, no other studies have accounted for this association (Jack et al., 1999). These contradictory findings may be attributed to several factors including the type of population studied, diagnostic criteria of cognitive impairment used or length of followup. Several lines of evidence suggest that the ␣-1antichymotripsin (ACT) gene is likewise another possible candidate gene for dementia susceptibility. ACT is a member of the serine protease inhibitor family and an acute phase reactant protein (Travis and Salvesen, 1983). It has been immunohistochemically identified as a component of the senile plaques associated with AD (Abraham et al., 1990). Reactive brain astrocytes around the amyloid plaques express increased ACT mRNA (Abraham, 2001). In vitro studies have shown that ACT can recognize and interact with the amyloid peptide A␤1−42 and stimulates assembly of A␤ into filaments that could facilitate its deposit in a concentration-dependent manner (Eriksson et al., 1995). These data have been also confirmed in transgenic mice (Nilsson et al., 2001). Furthermore, elevated serum and CSF concentrations in AD patients have been frequently described (Licastro et al., 1995). Kamboh et al. (1995) were the first to report that the Ala/Thr (A/T) polymorphism located at the signal peptide of the ACT gene conferred a significant risk for AD and modifies the risk associated with the ␧4 allele on the APOE gene (APOE4). This polymorphism is in strong linkage disequilibrium with a polymorphism (G → T) at position −51 in the promoter region of the gene and it has been shown that both may affect the expression and secretion of ACT (Morgan et al., 2001). Patients with probable AD carrying the ACT TT genotype in the signal peptide or in the promoter presented the highest plasma ACT levels and it has been shown that this genotype increases the risk of AD and is also related with a more rapid decline in patients with the APOE4 allele (Licastro et al., 2005). Nevertheless, other studies have not found an elevation of serum ACT concentrations nor association between

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the ACT gene and AD (Muller et al., 1996; Murphy et al., 1997). As a result of the so-called “cholinergic hypothesis” of AD, genes from brain cholinergic pathways could be another source of candidate genes for cognitive decline. One of the earliest neuropathological abnormalities in AD is the degeneration of cholinergic neurons located in the basal forebrain. Post mortem and antemortem studies in aged humans and AD patients have reported a large number of cholinergic abnormalities including decreases in choline acetyltransferase activity, acetylcholine release, nicotinic and muscarinic receptor expression, high-affinity choline uptake or dysfunctional neurotrophin support that positively correlate with cognitive decline and non-cognitive behavioural disturbances as well as with the deposition of toxic neuritic plaques (Auld et al., 2002). Recently, several studies have demonstrated that the toxic amyloid beta peptide (1–42) (Ab42) binds with high affinity to the nicotinic receptor ␣7 (␣7nAChR) on neuronal cell surfaces and that this binding may be a precipitating event in the formation of amyloid plaques in AD (Wang et al., 2000). It has been shown that the Ab1–42/␣7nAChR complexes internalize and accumulate inside lysosomal compartments causing a subsequent neuronal lysis and the formation of amyloid plaques from remnants of the degenerated neurons. Conversely, cells that do not express ␣7nAChR show no significant Ab42 binding (D’Andrea and Nagele, 2006; Wang et al., 2000). Researchers have suggested that this mechanism may be responsible for the selective vulnerability of cholinergic neurons that express the ␣7nAChR in AD brains. Leonard et al. (2002) have described a functional C → T polymorphism in the core promoter region (−86 C/T) of the ␣7 neuronal acetylcholine receptor subunit gene (CHRNA7) that decreases luciferase transcription by 20%. Considering that the degree and extent of Ab42 internalization was directly related to the number of ␣7nAChR expressed (Nagele et al., 2002), the presence of this variant may have an influence in the severity or the rate of progression to dementia. The aim of this study was to identify genetic markers that may predispose to dementia in patients with amnestic MCI. For this purpose, we investigated if the −86 C/T promoter polymorphism in CHRNA7 gene, the signal peptide polymorphism of ACT gene or APOE genotype are associated with an increase risk of amnestic MCI or have an effect on the risk of evolution to dementia in this population.

2. Methods 2.1. Subjects We performed a longitudinal prospective study on 89 subjects (30 male, 59 female) with mean age 75 (S.D. 7.1) who were admitted to the Memory Unit within the Geriatrics and Neurology Departments at Hospital Clinico San Carlos (Madrid, Spain). All of them fulfilled the Petersen criteria

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for amnestic MCI (Petersen et al., 2001): (1) memory complaint, preferably corroborated by an informant, (2) impaired memory function for age and education, (3) preserved general cognitive function, (4) intact activities of daily living, (4) not demented. These criteria correspond to stages 2 and 3 of the Reisberg Global Deterioration Scale (GDS). Patients were selected by opportunity, as they were consecutively admitted to the Geriatric and Neurology department between May 1999 and August 2005. We primarily included 130 individuals who attended the Departments of Neurology and Geriatrics complaining for memory problems and with no difficulties on activities of daily living. We excluded subjects who met dementia criteria as per DSM-IV or ICD-10, with functional or social disabilities, with a GDS score higher than 3, with more than four points in the Hachinski Ischaemia Scale, with history of stroke, toxic abuse, metabolic, endocrine, inflammatory, infectious, terminal, renal, hepatic or current psychiatric diseases, with uncertain development or very low education level. Additional investigations on every subject (Class II evidence level) as recommended by the American Neurology Association were completed. Then, six individuals were excluded for a GDS score higher than 3, seven for presenting a cognitive impairment not only in memory but in other cognitive domains, six for a Mini Mental State Examination (MMSE) score under 25, and 18 subjects were excluded by their medical history or analytical findings. Finally, other four patients were excluded because on a brief period of time, three showed deficits in other cognitive domains and developed other forms of dementia and one subject experienced a memory improvement and was considered as a normal aging subject. Follow-up study was carried out in the remainder 89 subjects. Two of them did not give their consent for the genetic study. To assess the risk associated to each genotype a case–control study was carried out. For this purpose, a control group of 90 healthy volunteer subjects (34 male, 56 female), selected from a day care institution, with mean age 76.08 (S.D. 9.7) without cognitive impairment (Mini Mental State Examination score ≥28 and without memory complains) was included. The study was performed in accordance with both the international ethical recommendations regarding research and human clinical trials as set out in the Helsinki Declaration (1964) and the guidelines for good clinical research practice as dictated by the World Health Organisation (1995). The procedure of the research project was explained to the subjects participating in the study and informed consent was obtained from each subject. 2.2. Clinical assessment All patients underwent a clinical evaluation. Age, gender, education level, social support and professional status of the patient were assessed. Cognitive assessment MCI was supported on the Mini Mental State Examination (Folstein et

al., 1975) GDS and the following neuropsychological tests: for cognitive domain cognitive subscale (CAMCOG) of the CAMDEX, Rivermead Barrage Memory Test, Benton visual memory test and Wechsler Adult Intelligence Scale; for functional domain Blessed Dementia Scale and Lawton and Brody activities Scale; and for affective domain Yesavage Geriatric Depression Scale. A detailed description of the cognitive domains assessed and the subscales used was recently published by our group (Marcos et al., 2006). 2.3. Clinical follow-up All 89 patients were followed up for a median time of 49 months (IQR, 38–55) with symptomatic and cognitive assessment every 6 months. After this period, some patients developed dementia while others retained the initial diagnosis of amnestic MCI. Patients were divided into two groups: Group I: 62 patients who developed dementia according to the DSM-IV criteria or who showed stages 4–7 on the GDS. The appropriate classification of dementia as AD was based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCS-ADRDA) criteria, a score in the Hachinski Ischemia Scale lower than 5, with no evidence of cerebral cortical infarcts and no more than a subcortical lacunar infarct lower than 1.5 cm on computed tomography (CT) or magnetic resonance imaging (MRI). We defined these patients as having progressive amnestic MCI (PMCI). Group II: 27 patients who maintained the initial diagnosis of amnestic MCI. These subjects are defined as having stable amnestic MCI (SMCI). Patients of this group remained on stages 2 and 3 of the GDS. Neuropsychological data on 82 patients and cognitive differences between the two groups of evolution were recently published (Marcos et al., 2006). 2.4. Genetic study Control group and the 87 patients gave their consent for genetic study. Genomic DNA was extracted from EDTAanticoagulated whole blood samples using standard DNA isolation methods. APOE and ACT genotyping was performed using polymerase chain reaction–restriction length polymorphism (PCR–RFLP) assays. Detection of APOE genotype was done using a method adapted from Zivelin et al. (1997) but using the forward primer from the method described by Hixson and Vernier (1990) that yields a 227 bp DNA fragment. Double digestion of this fragment with AflIII and HaeII yields on 4% agarose electrophoresis three specific fragments: 195 bp for APOE E4 allele, 177 bp for E2 allele and 145 bp for E3 allele respectively. ACT genotyping was performed according to the protocol previously described by

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Kamboh et al. (1995). CHRNA7 polymorphism was genotyped through TaqMan technology on an ABI7900 system (Applied Biosystems). A standard PCR reaction of 5 ␮L were carried out using TaqMan Universal PCR Master Mix and primers and probes designed by the Assay-by-Design service of Applied Biosystems. The probes used were 5 - FAM: CACCTCACCGCTCG - 3 and 5 - VIC: CACCTCGCCGCTCG - 3 and used primers were 5 - GCGCCCGGCTCCTTAAA 3 (forward) and 5 - GCCTGCGGCCACAGA - 3 (reverse). 2.5. Statistical analysis Comparisons of genotype frequencies were performed according the ␹2 analysis. Logistic regression analysis was done to calculate the odds ratios (ORs) and associated confidential intervals (CI) for amnestic MCI. Hardy–Weinberg (H–W) equilibrium was determined by the ␹2 exact test. Kaplan–Meier survival-analysis method and Cox proportional hazard analyses with dementia used as event were performed to study the effect of each genotype in the MCI progression to dementia. With the purpose to differentiate or remark the effect of any genotype in the RPMCI group (defined hereinafter in the paragraph of results), Kaplan–Meier survival analysis and proportionality of hazards over time for the RPMCI group compared with the combined SPMCI and SMCI group was assessed. We accomplished a Cox regression model to assess predictive factors of progression as compared to stable patients and factors of rapid progression as compared to the combined group of slow progressive patients and stable patients. In the hypothesis contrast, the null hypothesis was rejected with a type 1 or α error of <0.05. Data were analyzed with SPSS 12.0 for Windows and Epidat 2.01.

Fig. 1. Predictive cumulative hazard of developing Alzheimer’s disease from mild cognitive impairment. *Median length of follow-up of the cohort = 49 months (IQR, 38–55). **Median disease free survival time = 24 months. ***Benchmark time of slope switching = 20 months, cumulative survival at that time = 0.56.

3. Results Survival curve is shown in Fig. 1. The shape of the curve revealed two different slopes that intersect at the benchmark of month 20. This cut off point allowed us to distinguish

Table 1 Baseline sociodemographic characteristics and genotype data of the stable and progressive MCI patients Variable (years)a

Age Age range (years) Male (%)b Duration of disease evolution (months)a Length of schoolingb ≤10 years (%) Professional statusb (% unqualified) Social supportb (% with low rating)

PMCI n = 62

SMCI n = 27

p-value

77.0 (74.0–80.2) 53–92 22 (35.5) 24.0 (18.0–36.0) 54 (87.1) 43 (72.9) 11 (19.6)

74.0 (65.0–77.0) 53–80 8 (29.6) 22.0 (12.0–36.0) 22 (81.5) 19 (70.4) 5 (20.8)

0.008 0.58 0.45 0.49 0.81 0.90

Genotype Ala/Thr ACTb

AA AT TT

15 (25.00) 31 (51.67) 14 (23.33)

9 (33.33) 12 (44.44) 6 (22.22)

0.71

CHRNA7b

−86 CC −86 CT

53 (91.38) 5 (8.62)

26 (96.30) 1 (3.70)

0.38

APOEb

33 23 34 44

30 (50.00) 1 (1.67) 27 (45.00) 2 (3.33)

16 (59.26) 2 (7.41) 7(25.93) 2 (7.41)

0.22

PMCI = progressive mild cognitive impairment; SMCI = stable mild cognitive impairment. a Median (IQR) (median test). b Absolute number (%) (␹2 or Fisher).

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Table 2 Baseline sociodemographic characteristics and genotype data of rapid progressive MCI patients and combined slow progressive with stable MCI Variable (years)a

Age Age range (years) Male (%)b Duration of disease evolution (months)a Length of schoolingb ≤10 years (%) Professional statusb (% unqualified) Social supportb (% with low rating)

RPMCI n = 40

SMCI + SPMCI n = 49

p-value

78.0 (74.2–80.7) 60–92 12 (30.0) 24.0 (20.2–36.0) 38 (95.0) 29 (76.3) 6 (17.6)

75.0 (69.5–78.0) 53–84 18 (36.7) 22.0 (12.0–36.0) 38 (77.6) 33 (68.8) 10 (21.7)

0.09 0.50 0.15 0.01 0.43 0.65

Genotype Ala/Thr ACTb

AA AT TT

7 (17.50) 24 (60.00) 9 (22.50)

17 (36.17) 19 (40.43) 11 (23.40)

0.10

CHRNA7b

−86 CC −86 CT

37 (94.87) 2 (5.13)

42 (91.30) 4 (8.70)

0.51

APOEb

33 23 34 44

22 (55.00) 0 (0.00) 17 (42.50) 1 (2.50)

24 (51.06) 3 (6.38) 17 (32.17) 3 (6.38)

0.398

RPMCI, rapid progressive mild cognitive impairment; SMCI, stable mild cognitive impairment; SPMCI, slow progressive mild cognitive impairment. a Median (IQR) (median test). b Absolute number (%) (␹2 or Fisher).

Table 3 shows the frequencies in amnestic MCI patients and controls of the APOE, ACT and CHRNA7 genotypes or combined genotypes taking as reference the more frequent genotype in control group. As has been done by others authors, APOE genotypes were divided either into E4 positive (E4E4, E4E3, E4E2), E2 positive (E2E3 and E2E2) and E3 homozygotes (E3E3). There were no subjects in the group of patients with the E2E2 genotype. As some evidence suggests that E2 may be protective, two control subjects with the E2E4 genotype were included in the E4 positive group, after analyzing that the deleterious effect of E4 was higher than the protective effect of E2 when E2E4 subjects were alternatively included o excluded in any of the groups. For ACT polymorphism, AA genotype was the most representative in control group, so we grouped subjects as T carriers, that comprise combined TT and AT genotypes, and T non-carriers. We did not find any subject TT homozygous for CHRNA7

two separated subgroups of PMCI patients: 40 patients that converted to AD before 20 months of evolution – therefore we defined them as rapid progressive amnestic MCI (RPMCI) –, and 22 that changed the diagnostic after 20 months that we defined as slow progressive amnestic MCI (SPMCI) Sociodemographic and genotype data for PMCI and SMCI groups are shown in Table 1 and for RPMCI and combined SPMCI + SMCI groups in Table 2. There were no significant differences between them in sex distribution or in length of disease evolution. No relevant differences were noted between groups with respect to professional status, social support or APOE, ACT and CHRNA7 genotype frequencies. We found statistical difference in years of schooling between RPMCI and SPMC + SMCI. Age was also significant different in all groups of comparison. Two patients refused to participate in the genetic study but continue the clinical and neuropsychological study.

Table 3 Genotype distribution of the APOE, ACT and CHRNA7 polymorphism in patients with MCI and controls and Odds Ratios for amnestic MCI Genotype

Controls n

APOE

Ala/Thr ACT CHRNA7

Cases %

n

OR

CI 95%

p- value

%

33 22 + 23 24 + 34 + 44

70 8 12

77.8 8.9 13.3

46 3 38

52.9 3.4 43.7

1 0.57 6.04

0.14–2.26 2.76–13.23

0.409 <0.001

AA AT + TT

26 64

28.9 71.1

24 63

27.6 72.4

1 1.07

0.55–2.05

0.847

−86 CC −86 CT

82 5

91.1 5.6

73 6

83.9 6.9

1 1.3

0.38–4.43

0.632

OR, odds ratio; CI, confidence interval. APOE genotypes were divided either into E4 positive, E2 positive and E3 homozygotes. For ACT polymorphism, subjects were divided as T carriers and T non-carriers. We did not find any subject TT homozygous for CHRNA7 polymorphism.

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Fig. 2. Effect of APOE, ACT and CHRNA7 genotypes on survival time (Kaplan–Meier curve) to AD in patients with MCI. The endpoint event was development of dementia. (a) Comparison of APOE 33 genotype with 23 genotype ([HR] = 0.4, 95% CI = 0.05–2.7, p = 0.25) and with combined 34 + 44 genotypes ([HR] = 1.2, 95% CI = 0.73–2.1, p = 0.32) (Cox model). (b) Comparison of CHRNA7 −86 CC and −86 CT genotypes ([HR] = 0.5, 95% CI = 0.2–1.5, p = 0.1) (Cox model). (c) Comparison of ACT AA and AT + TT genotypes ([HR] = 1.3, 95% CI = 0.7–2.3, p = 0.4) (Cox model).

polymorphism. The distribution of all genotypes was in H–W equilibrium in control group. The E4 positive genotype was more represented in patients than in controls and conferred a significant increased odds ratio for amnestic MCI (odds ratio [OR] = 6.04, 95% confidence interval (CI) = 2.76–13.23, p < 0.001). No statistically significant difference in genotype frequencies between both groups was observed for ACT and CHRNA7 polymorphisms (for AT + TT genotype: [OR] = 1.07, 95% CI = 0.55–2.05, p = 0.84; for −86 CT genotype: [OR] = 1.3, 95% CI = 0.38–4.43, p = 0.67). Kaplan–Meier survival analysis of PMCI group compared with SMCI group and Cox proportional hazard regression analyses have shown that APOE, ACT nor CHRNA7 genotypes predict the conversion to AD; therefore, there is a trend for −86 CT CHRNA7 genotype to show a protective effect (hazard ratio [HR] = 0.5, 95% CI = 0.2–1.5, p = 0.1) (Fig. 2).

Comparison of RPMCI group with the combined SPMCI + SMCI group shows that the presence of T allele of ACT conferred a nearly statistical significant risk to evolve to dementia before 20 months ([HR] = 2.03, 95% CI = 1–4.6, p = 0.06) (Fig. 3). Although it is not statistical significant, we think that it is an important data, because with estimations higher than 1.8 – considered as the residual confounding value – it is not probable that an unknown variable could have an effect on this relation and therefore might change the significance of this effect in observational studies. To evaluate the predictive significance of these genotype data, a Cox regression model was done including the variables age and years of schooling that were statistically different between groups (Table 4). ACT genotype, age and years of schooling are significant predictive factors of evolution to AD before 20 months. Age and years of schooling do not confound the genetic effect; therefore ACT genotype

Fig. 3. Effect of APOE, ACT and CHRNA7 genotypes on survival time (Kaplan–Meier curve) to AD in patients with MCI. The endpoint event was development of dementia before 20 months. (a) Comparison of APOE 33 genotype with 23 genotype ([HR] = indefinite, p = 0.1) and with combined 34 + 44 genotypes ([HR] = 1.1, 95% CI = 0.6–2.1, p = 0.5) (Cox model). (b) Comparison of CHRNA7 −86 CC and −86 CT genotypes ([HR] = 0.5, 95% CI = 0.1–2.1, p = 0.3) (Cox model). (c) Comparison of ACT AA and AT + TT genotypes ([HR] = 2.03, 95% CI = 1–4.6, p = 0.06) (Cox model).

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Table 4 Cox regression model to evaluate the predictive variables of evolution to AD before 20 months Variable

Exp (B) (HR)

CI 95%

p-value

Age Length of schooling Ala/Thr ACT −86 C/T CHRNA7 APOE

1.09 0.23 2.87 0.39 1.27

1.03–1.15 0.05–0.98 1.24–6.64 0.09–1.70 0.91–1. 77

0.001 0.016 0.007 0.156 0.171

HR, hazards ratio; CI, confidence interval.

is the variable that confers the highest risk of rapid evolution to dementia. Years of schooling and CHRNA7 appears as significant or a trend of significant respectively variables of protection. APOE genotype is not a predictive factor of progression to AD.

4. Discussion One of the most important challenges of investigation in MCI is to identify genes that could be used to predict which subjects will progress to dementia. Individuals genetically predisposed to evolve to dementia could be benefited of a treatment in an early stage of the disease in which the neurodegeneration has not progressed, delaying their evolution a limited number of years, what would improve their quality of life and that of their relatives. In addition, genetic studies of MCI considering their evolution could furthermore explain the diagnostic heterogeneity of this entity. In this sense, many authors defend that the nosological classification of MCI demands not only to define its clinical characteristics, but also its aetiology (Dubois and Albert, 2004). As has already been noted by some authors (van Duijn, 2004; Winblad et al., 2004), the heterogeneity of MCI has inferred one of the difficulties in the genetic investigation. In one hand, probably each one of the diseases that underlie the diagnosis of MCI may in part have a genetic origin, thus it may be different genes for different aetiologies of MCI and in the other hand, most of these pathologies are common diseases with complex genetic inheritance, that means that are a result of the interaction of multiple genes and the environment and that are characterized for presenting genotypes with incomplete penetrance and a low magnitude associated risk. In these cases, candidate genes studies represent the best approach to identify involved genes in their aetiology and, for some authors, the use of a combination of genetic tests, the most appropriate method for predictive studies (Yang et al., 2003). Following Petersen’s recommendations to diminish the effect of heterogeneity all the subjects included in our study met criteria for amnestic MCI (Petersen et al., 2001). It has been well established that dementia risk varied with the definition of MCI used and that the highest risk for dementia of AD type was observed for amnestic MCI. Some authors have demonstrated that up to 80% of subjects with amnestic

MCI convert to AD in approximately 6 years, that there is a 100% dementia risk in subjects with amnestic MCI older than 70 years in ten years of follow-up and that most of these patients have neuropathological changes of AD and share similar biomarkers (Jicha et al., 2006; Petersen et al., 2001, 2006; Visser et al., 2006) Taking this into account, we have selected as candidate genes in our study, genes that have already been implicated in the susceptibility to develop AD and/or are involved in the formation of amyloid plaques. We have found that the cumulative percentage of patients with amnestic MCI dropped out at 20 months so the proportion of patients who develop dementia was much higher before that time. Taking into account this evolutionary pattern, we have separated the patients in different groups according to the rapidity of its diagnostic change to AD, hypothesizing that each group could be genetically different or be modulated in its evolution for different genes. We have found a similar pattern of evolution than several populationbased studies (Johansson et al., 1992; Palmer et al., 2002). All of them reported that the progression from MCI to dementia is time dependent and occurs mostly within the initial 2–3 years of observation. In accordance with our results, recently Busse et al. (2006) have published that patients with similar diagnostic criteria of MCI that in our study had the highest conversion rates (20%) during the first 18 months of the study. Our results demonstrate that the presence of the APOE4 allele is a risk factor for the development of amnestic MCI, but once MCI is established the APOE status do not predict the conversion to AD. In our sample, individuals carrying at least one allele 4 have a six more time a risk to develop amnestic MCI than E3 homozygote. These data are in agreement with other positive association studies (Engelborghs et al., 2003; Tschanz et al., 2006; Zill et al., 2001) in which it has been described a nearly six-fold higher risk to MCI associated to E4 allele and particularly replicate the results inferred from Spanish population by other groups (BartresFaz et al., 2001; Coria et al., 1995; Rosich-Estrago et al., 2004). Furthermore, we have observed that if we grouped data from subset of MCI patients from different studies, in most cases E4 allele is overrepresented (Herukka et al., 2006; Lavados et al., 2005). Not only case–control studies, but in other prospective clinical studies that estimate the incidence of MCI among cognitively healthy subjects, support that the APOE4 allele independently increases the risk for MCI (Tervo et al., 2004). Nevertheless, other authors have not demonstrated the association between MCI and genotyping, possibly because insufficient study power (Katzman et al., 1997; Lautenschlager et al., 2005). Furthermore, a considerable amount of studies have been published examining the role of APOE genotyping in the conversion from MCI to dementia since Petersen et al. (1995, 1996) reported that APOE4 status could be a predictor of clinical progression to AD. Nevertheless, although it is true that results have been diverse and contradictory, most of the studies showed that APOE4 status is not a reliable predictor

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of decline to AD once MCI is established, as occurs in ours. The same group mentioned above, in a posterior research paper in which they included the hippocampal volume into a bivariate model using APOE and other variables, concluded that only hippocampal atrophy is predictive of subsequent conversion to AD in MCI patients (Jack et al., 1999). A lot of studies have also reported similar negative results (Amieva et al., 2004; Arnaiz et al., 2004; Devanand et al., 2005; Drzezga et al., 2005). For some authors, the negative results are a consequence of the limited number of patients included in the studies, the short time of follow-up, the insufficient control from potentially confounding factors and the lack of consensus criteria for MCI (Aggarwal et al., 2005). Without underestimating the first three, we think that definition of MCI is one of the most important sources of variability in this type of studies. Arnaiz et al. (2004) have compared two different samples from two different institutions, one with amnestic MCI and the other with multiple domains MCI and have analyzed possible predictors for the conversion from MCI to AD. When all the patients were considered together in a Cox proportional hazard model only the number of affected cognitive factors was a significant predictor of time to AD, but age, number years of education and APOE4 genotype were not. When the same Cox analysis was conducted for the two institutions separately, the effect of APOE4 genotype remained not significant in the amnestic group but became a significant predictor for the multiple domains in the MCI group (Arnaiz et al., 2004). In agreement with other authors, we consider that this occurs because amnestic MCI is a more homogeneous entity and actually represents an early stage of AD, therefore the APOE genotype in fact marks the risk of AD. Hence, some recently positive studies were described from a sample that fulfilled criteria of cognitive impairment but not dementia (CIND) which are considered the most broad-based criteria of MCI and therefore composed of a more etiologically heterogeneous group (Davis and Rockwood, 2004; Feldman and Jacova, 2005). Recently, Kryscio et al. (2006), using a polytomous logistic model to represent transitions among different cognitive states over time from normal, amnestic or mixed MCI, dementia or death have shown that the presence of at least one APOE4 allele affects transitions into amnestic MCI from the normal state and to lesser extent, transitioning to mixed MCI. Nevertheless the APOE4 allele does not significantly increase the odds of a conversion from amnestic or mixed MCI to dementia except in cases where the person moves from the cognitively normal state to dementia without going through a detectable MCI state experiencing a very rapid deterioration in a one year period (Kryscio et al., 2006). Although the presence of patients carrying APOE2 allele is insufficient to make any meaningful comparisons regarding the predictive role of Apoe2 allele in progression of MCI to AD, we think that it is interesting to mention that no patient of our study carrying this allele evolved to dementia in less than 20 months. There are some reported data about the possible

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protective role of APOE2 against AD although the results are conflicting (Corder et al., 1994; Talbot et al., 1994). In relation to the – 17A/T signal peptide polymorphism and the – 86 C/T polymorphism of the CHRNA7 promoter region the contrary phenomenon occurs to that with the APOE polymorphism: CHRNA7 polymorphism does not confer risk to developing amnestic MCI, but modifies the risk to evolve AD in time in an opposite manner. The presence of at least one copy of the T allele of the ACT polymorphism is associated with a risk of evolving dementia rapidly. These results, in some aspects, are in conformity with those published by Licastro et al. (2005). They have found that TT genotype of a polymorphism in the promoter region of the ACT gene was more represented between patients with probable AD of early onset than in a similar control population, independently of the effect of the APOE4 allele. The T allele in the promoter region is in almost complete linkage disequilibrium with the T allele in the signal peptide region of the ACT gene (Licastro et al., 2005). Furthermore, this genotype was more frequent in patients that deteriorated faster, that is, that reduced their punctuation in the MMSE by more than five points per year. Recently, it has been reported that an association between high levels of serum ACT and a high risk of decline in MMSE punctuation, adjusted by age, sex and educational level among a cohort of older persons aged 62–85 years (Dik et al., 2005). Presence of the T allele of −86 C/T CHRNA7 polymorphism was associated with a reduction of 50% in the probability of progressing to AD in 5 years. Taking into account that the T variant is associated with a decrease in the transcription of the CHRNA7 gene (Leonard et al., 2002) the presence of a reduction in the number of CHRNA7 receptors could delay the evolution to AD. These results are in agreement with a number of recent studies in which it has been demonstrated that the Abeta42 is first accumulated inside vulnerable neurons provoking its lysis before the formation of the amyloid plaque. The most vulnerable neurons are those that express more CHRNA7 receptors as a consequence of their high-affinity binding to Abeta42 on cell surfaces, endocytosis of this complex and degeneration of the overburden neurons. This mechanism may explain the selective vulnerability of cholinergic neurons in AD brains (Nagele et al., 2002). It has been found that intact ␣7 nicotinic acetylcholine receptor protein levels in peripheral blood leukocytes are significantly higher in AD patients than in a normal control group and that are significantly inversely correlated with MMSE scores (Chu et al., 2005) and it has been recently found that post mortem brain binding for nicotinic acetylcholine receptors did not differ between a control sample and MCI patients but it was significantly reduced in a AD. Accordingly, with our results, nicotinic receptor binding seems to be lost during the transition from MCI to AD (Sabbagh et al., 2006). We have found that there are differences in age between all groups of progression to AD, in such way that RPMCI patients were older than SPMCI patients and at the same time this group was older than SMCI. Most previous studies

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support similarly that age is a powerful risk factor for the occurrence of sporadic AD. In the general population, the risk of AD beyond the age of 65 increases exponentially, doubling every 5 years, and at 85, the annual incidence of AD is 20-fold greater than at 65 (Bachman et al., 1993; Ott et al., 1998). In a recent study of the 10-year risk of dementia in subjects with MCI aged 40–85 years, the dementia risk in subjects with MCI was strongly dependent on age for all MCI definitions used and particularly higher for amnestic MCI. In all cases, younger subjects with MCI progressed to dementia at longer follow-up intervals than elderly subjects did. The authors supposed that this is because they were in an earlier stage of the disease or that the rate of progression of the neurodegenerative process were slower in younger subjects (Visser et al., 2006). For some other authors, the accumulation of biological changes produced while the brain ages is the fundamental major cause of neural and cognitive decline in the elderly and in some individuals to further transition from MCI to AD (Drachman, 2006). We have found that the percentage of patients that have completed less or equal than 10 years of schooling was significantly lower in the group of RPMCI than in the rest of patients. These data are in concordance with the recently established concept of cognitive reserve. This concept is based on the idea that cognitive engagement begins early in life and persists over time providing different cognitive reserve regardless brain pathology and modulating the clinical expression of AD pathology (Stern, 2006). The cognitive reserve hypothesis suggest that education may postpone the manifestation of dementia symptoms among individuals with AD pathology by allowing them to use cognitive processing or compensatory strategies to cope better with brain damage (Mortimer et al., 2005). Several studies have confirmed that individuals with higher education have a reduced risk of developing AD (Bennett et al., 2003; Lindsay et al., 2002). Years of education affect the occurrence and timing of dementia symptoms and it is predictive of dementia status among individuals with neuropathological AD. Individuals with greater cognitive reserve, as reflected in years of education, are better able to have greater brain pathology without observable deficits in cognition (Gatz et al., 2006; Roe et al., 2007). Despite the differences in age and in years of education between the groups of evolution to AD we have found with a Cox regression model that both variables not only do not confound the effect of the ACT or CHRNA7 genes, but, for example in the case of the ACT polymorphism, the presence of the variant confers significantly higher risk to evolve rapidly to AD than age and its effect is as robust as the protective effect of years of schooling. Our study has several limitations. We utilized a relatively small sample size. However, for example, in the case of −86C/T CHRNA7 polymorphism, given its low frequency in general population, we consider that the protective effect in the survival analysis is not valueless, even though its effect was not statistically significant after taking the other variables

into account in the Cox regression model. Additionally, conclusions about our findings are made from patients selected from a memory clinic; consequently they must be cautiously generalized to the population. A larger sample with a greater number of patients and from different sources of origin is needed to replicate our data. Therefore, we think that our results demonstrate that carrying at least one APOE4 allele is a risk factor to develop amnestic MCI, but once established, the gene does not have effect in the risk to evolve to AD. However, the ACT and CHRNA7 polymorphisms were not associated with amnestic MCI risk, but both seem to modify the risk of progression to dementia in opposing manners: ACT polymorphism increasing the risk to evolve to AD rapidly and CHRNA7 polymorphism protecting MCI patients from evolution to dementia.

Conflicts of interest All authors disclose that there is no actual or potential conflict of interest that could inappropriately influence their work.

Acknowledgement The research was partially supported by a grant from the Comunidad de Madrid, reference number 08.5/0009/1998, Spain.

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