Journal of Alzheimer’s Disease 32 (2012) 919–926 DOI 10.3233/JAD-2012-120358 IOS Press
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Influence of COMT Val158Met Polymorphism on Alzheimer’s Disease and Mild Cognitive Impairment in Italian Patients Cristina Lannia,1,∗ , Giulia Garbinb,1 , Antonella Lisac , Fabrizio Biundoa , Alberto Ranzenigod , Elena Sinforianie , Giovanni Cuzzonif , Stefano Govonia , Guglielmina Nadia Ranzanib and Marco Racchia a Department
of Drug Sciences, Centre of Excellence in Applied Biology, University of Pavia, Pavia, Italy of Biology and Biotechnology, University of Pavia, Pavia, Italy c Institute of Molecular Genetics, CNR, Pavia, Italy d Department of Geriatric Medicine, Ospedale S. Orsola Fatebenefratelli, Brescia, Italy e Alzheimer’s Disease Assessment Unit/Laboratory of Neuropsychology, IRCCS “C. Mondino, National Institute of Neurology” Foundation, Pavia, Italy f A.S.P.S. Margherita Geriatric Department, Pavia, Italy b Department
Handling Associate Editor: Benedetta Nacmias
Accepted 29 June 2012
Abstract. COMT (Catechol-O methyltransferase) gene is one of the key players in synaptic plasticity and in learning and memory mechanisms. A single nucleotide polymorphism (rs4680; G to A) in the COMT coding region causes Val158Met aminoacid substitution in the corresponding protein, with Val allele exhibiting a 3- to 4-fold increase in enzyme activity compared to Met. With the purpose of examining the influence of COMT as a genetic risk factor for cognitive impairment, we analyzed a sample of 248 healthy subjects, 276 patients affected by Alzheimer’s disease (AD), and 70 subjects with mild cognitive impairment (MCI), the latter condition possibly representing a prodrome for dementia. All subjects were analyzed for COMT rs4680 polymorphism and APOE genotype. Our study strengthens data showing that APOE ε4 allele is an independent risk factor for AD and also a risk factor for MCI. Neither COMT alleles nor genotypes proved to be independently associated with the risk of AD or MCI in our sample. However, we found an association between COMT GG genotype (Val/Val) and APOE ε4 carrier status and the risk of AD and MCI. In particular, when GG genotype is included into the multinomial analysis, the risk of AD and MCI due to APOE ε4 allele is increased to about 2-3 fold; moreover, the risk conferred by the combination of G and ε4 alleles is more pronounced in male patients. To our knowledge, this synergistic effect is here shown for the first time on a population sample representative of Caucasian patients. Keywords: Alzheimer’s disease, apolipoprotein E, catechol-O methyltransferase (COMT), mild cognitive impairment, polymorphism, risk factor
1 These
authors contributed equally. to: Cristina Lanni, PhD, Department of Drug Sciences, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy. Tel.: +39 0382 987396; Fax: +39 0382 987405; E-mail:
[email protected]. ∗ Correspondence
ISSN 1387-2877/12/$27.50 © 2012 – IOS Press and the authors. All rights reserved
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C. Lanni et al. / Effect of COMT Variants in AD and MCI
INTRODUCTION Alzheimer’s disease (AD) is a polygenic multifactorial disease characterized by the interaction of environmental, metabolic, and genetic factors. The analysis of both familial and sporadic AD cases, either late or early onset, revealed the involvement of several genes. Rare disease-causing mutations in AβPP (amyloid- protein precursor), PSEN1 (presenilin 1), and PSEN2 (presenilin 2) genes have been associated with early-onset familial disease, while APOE (apolipoprotein E) ε4 allele has been recognized as the single most important risk factor for late-onset disease [1]. Recent genome-wide association studies (GWAS) have delivered a great number of potential AD susceptibility alleles that are common in the general population; however, the identified loci exert very small risk effects and only explain a fraction of the genetic risk [2]. Moreover, it is conceivable that additional genes might modulate the effect of susceptibility variants including APOE ε4 allele, to initiate the pathogenesis of AD. A possible modulator is COMT (Catechol-O methyltransferase), the product of which catalyzes the degradation of catecholamines and the methylation of catechol estrogens [3]. The interest on COMT in neurodegenerative diseases characterized by cognitive impairment is based on recent findings that the protein likely contributes to cognitive functions due to its role in dopamine degradation [4]. Indeed, agerelated loss of dopaminergic function has been reported to correlate with cognitive impairment [5]: dopamine is necessary for prefrontal cortex (PFC) function and dopamine imbalance may produce deficits in PFCdependent tasks [6]. The single nucleotide polymorphism (SNP) of COMT gene (rs4680 SNP; G > A) has been reported to have a functional significance: A allele (corresponding to methionine at codon 158) is associated with low protein activity compared to G allele (corresponding to valine) [7]. Cross-sectional studies showed that the Val/Val coding genotype is associated with less efficient cognitive processing and poorer performance in cognitive tests, compared to the Met/Met [8, 9]; however, this association has not been confirmed in recent longitudinal studies [10, 11]. COMT has been suggested as a candidate for susceptibility to AD-related psychosis (AD-P), and an association between ADP and polymorphic variants has been reported [12, 13]. By investigating Taiwan patients, Wang et al. [14] found that GG genotype (Val/Val) and APOE ε4 allele exert a synergistic effect upon the risk of AD. More recently, a study on Basque population indicated that
high-activity genotypes (GG and AG) do not represent an independent risk factor for AD or mild cognitive impairment (MCI), but exert a synergistic effect with APOE ε4 allele, being this effect increased in AD women [15]. With the aim of testing the influence of COMT as a risk factor for cognitive impairment, we genotyped control subjects and patients with AD or MCI, a condition possibly representing a prodrome for dementia. All subjects were analyzed for COMT rs4680 polymorphism and APOE genotype. We evaluated whether: (i) COMT is associated with risk of AD, (ii) there is an interaction with APOE, (iii) such interaction influences the risk of AD, (iv) COMT can affect parameters linked to the disease such as the age of onset in AD, and (v) COMT polymorphism can influence the rate of Mini Mental State Evaluation (MMSE) change over time to AD in MCI patients. All samples analyzed were from the Northern Italian population; due to genetic peculiarity of Basques and Taiwaneses, this study is the first one addressing the above questions by investigating a population sample representative of Caucasians. MATERIALS AND METHODS Subjects We analyzed 276 patients with sporadic AD, 70 patients with MCI, and 248 healthy age-matched controls (Table 1). Venous blood samples were obtained from the Institute “Fondazione Casimiro Mondino” and “Santa Margherita” in Pavia and from Sant’Orsola Hospital in Brescia, Northern Italy. The Ethical Committee approved the protocol of the study, including the follow-up visits, and a written consent was obtained from all subjects or, where appropriate, their caregivers. The study was done in accord with the ethical standards of the Committee on Human Table 1 Demographic and clinical variables of the analyzed subjects. n, number; M, male; F, female; LOI, length of illness; MMSE, Mini Mental State Examination. Data are expressed as mean ± standard deviation. LOI represents the time (months) elapsed from the clinical evaluation at the first visit n (M;F) Age LOI MMSE a p < 0.001
Controls
AD
MCI
248 (106;142) 74 ± 10
276 (90;186) 77 ± 8 44 ± 29a 15 ± 8b
70 (24;46) 71 ± 10 27 ± 12 25 ± 3c
28 ± 2
LOI, AD versus MCI. b p < 0.001 MMSE, AD versus control. c p < 0.001 MMSE, MCI versus control; t-test.
C. Lanni et al. / Effect of COMT Variants in AD and MCI
Experimentation of the Institutions participating to this research and was conducted in accordance with the Declaration of Helsinki concerning medical research in human subjects. A senior neurologist examined all the subjects and diagnosis of dementia was made according to DSM-IV and NINCDS-ADRDA criteria [16]. All MCI subjects met the original Petersen/Mayo criteria for MCI [17, 18]. Dementia was diagnosed upon interview, objective and neurological examination, cognitive evaluation, laboratory and radiological (CT Scan and MRI) investigation. Neuropsychological assessment was carried out by expert neuropsychologists, and was designed to evaluate: global cognitive function (MMSE) [19]; verbal (Verbal Span, Digit Span) and spatial (Corsi’s block-tapping test) short-term memory; verbal and visuo-spatial long-term memory (Logical Memory Test, the Rey Auditory Verbal Learning test, Rey’s Complex Figure B); logical-executive functions (Wisconsin Card Sorting test, Raven’s Matrices, Frontal Assessment Battery); visuospatial abilities (constructional apraxia, the Rey Complex Figure); semantic (animals, fruit, colors, cities) and phonological verbal fluency; and attention and visuo-perceptual speed (Attentive Matrices, Trail Making A and B) [20–24]. This battery of neuropsychological tests has been used only for diagnostic purposes, especially to evaluate the conversion from MCI to AD. Data from the neuropsychological parameters have not been included in our data analysis, since these were completed only for a subset of patients from all diagnostic groups. Control subjects were individuals with no clinical signs of neurological or psychiatric diseases, mostly enrolled among spouses of MCI and AD patients. None of the subjects selected in this study was affected by neoplastic or autoimmune disease when blood samples were taken. In order to evaluate the progression to AD, MCI subjects underwent follow-up visits every six months based on clinical interview, objective and neurological examination and neuropsychological evaluation; neuroimaging (CT or MRI) was performed every 12 months. The longitudinal observation lasted 36 months. Molecular genetic analysis Genomic DNA was extracted from peripheral leukocytes with a commercially available nucleic acid purification kit (Wizard DNA Purification Kit, Promega). The APOE gene polymorphisms were determined by Hha I restriction endonuclease digestion of PCR products, according to Hixson and Vernier
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[25]. For COMT polymorphism, the PCR reaction was carried out in a total volume of 25 L in a 0.2 mL reaction tube containing approximately 100 ng of DNA, 1× DreamTaq™ Green Buffer (20 mM TrisHCl pH 8.0, 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20 and 50% (v/v) glycerol) (Fermentas, Burlington, ON, Canada), 200 M of each dNTP, 0.4 M of each primer and 1 unit of DreamTaq™ Green DNA Polymerase (Fermentas, Burlington, ON, Canada). The PCR cycling parameters were: 1 cycle of 2 min at 98◦ C; 28 cycles of 1 min at 96◦ C, 30 s at 66◦ C for the annealing and 1 min at 68◦ C; 1 cycle of 5 min at 68◦ C. PCR was carried out using COMT primers; F: 5 -GATCCAAGTTCCCCTCTCTCCAC-3 and R: 5 -CACTGAGGGGCCTGGTGATAGT-3 . PCR products were directly sequenced to analyze the rs4680 SNP. Before sequencing, PCR products were enzymatically purified by using the ExoSAP-IT PCR Clean-up Kit (GE Healthcare, Amersham, UK). Purified products were sequenced with the same primers as for PCR, according to the Sanger procedure [26]; sequencing was performed by means of an automated DNA analyzer (Applied Biosystems, ABI PRISM 3700 capillary analyzer). Statistical analysis Differences among demographic and clinical variables were evaluated using the t-test. A dichotomous variable was used for each polymorphism: “1” or “0” for “carrier” or “non carrier” of the APOE ε4 allele and for different alleles and genotypes of COMT gene. The frequencies of APOE ε4 and COMT alleles in AD and MCI versus control subjects were compared using the standard χ2 test. The combined effects of APOE and COMT polymorphisms on AD risk were evaluated by multinomial logistic regression analysis, and odds ratios were calculated. When significance was found, a test for linear trend was also applied. Statistical analyses were performed by Stata, version 10 for Machintosh, (StataCorp. 2007; Stata Statistical Software: Release 10, College Station, TX: StataCorp LP.) and G*Power 3.1 [27, 28]. A p value < 0.05 was defined as statistically significant. RESULTS We analyzed a sample of 70 MCI patients, 276 AD patients, and 248 healthy subjects. The memory impairment evaluated by the MMSE showed a greater impairment in AD patients compared to MCI patients
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C. Lanni et al. / Effect of COMT Variants in AD and MCI
Table 2 Allele and genotype frequencies of APOE and COMT polymorphisms in controls, AD and MCI patients APOE Allele
Genotype
COMT Allele Genotype
Controls
AD
MCI
ε2 ε3 ε4 ε2/ε3 ε3/ε3 ε3/ε4 ε2/ε4 ε4/ε4
0.04 0.90 0.06 0.08 0.81 0.11 0 0
0.04 0.70 0.26 0.06 0.47 0.40 0.02 0.05
0.07 0.76 0.17 0.10 0.59 0.26 0.03 0.03
A G AA GA GG
0.50 0.50 0.23 0.53 0.24
0.47 0.53 0.22 0.51 0.27
0.49 0.51 0.24 0.49 0.27
Table 3 Exact G test for allele and genotype frequencies in controls (CS), AD and MCI patients APOE AD versus CS MCI versus CS AD versus MCI COMT AD versus CS MCI versus CS AD versus MCI
Alleles
Genotypes
p value
