JOURNAL OF JOURNAL OF

Volume 27 Number 2, 2005

ISSN: 0895–8696

JOURNAL OF

Molecular Neuroscience Editor-in-Chief: ILLANA GOZES, PhD In This Issue The Biological Clock Alzheimer’s Immunization Signal Transduction/ Neuroprotection/ Polymorphism

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Journal of Molecular Neuroscience Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/05/27:213–218/$30.00 DOI:10.1385/JMN:27:02:213

ORIGINAL ARTICLE

Monoamine Oxidase A Polymorphism in Brazilian Patients Risk Factor for Late-Onset Alzheimer’s Disease?

Agnes L. Nishimura,1 Camila Guindalini,1 João R. M. Oliveira,3 Ricardo Nitrini,2 Valéria S. Bahia,2 Paulo R. de Brito-Marques,3 Paulo A. Otto,1 and Mayana Zatz*,1 1

Human Genome Research Center, Biology Department, Institute of Biosciences, São Paulo University (IBUSP), São Paulo, Brazil; 2Neurology Department of Medical School, São Paulo University, São Paulo, Brazil; and 3Behavioral Neurology Unit, Department of Neurology, Faculty of Medical Sciences, University of Pernambuco, Recife, Brazil Received December 8, 2004; Accepted March 30, 2005

Abstract Different studies have attempted to find polymorphisms involved in the serotonergic pathway that could be involved in mood disorders and late-onset Alzheimer’s disease (LOAD) symptoms. Here, we compared the frequency of two polymorphisms: monoamine oxidase A (MAOA) and serotonin transporter in LOAD patients versus controls. No evidence of association was observed when these polymorphisms were compared separately; however, the combination of the MAOA allele 1 + the short allele of 5-HTTLPR + ApoE-ε4 was significantly more frequent in patients than in controls. It reinforces the hypothesis that different genes acting together might play a role in AD susceptibility. Based on these data, we suggest replicating these studies in larger samples of LOAD patients belonging to different ethnic groups. DOI:10.1385/JMN:27:02:213 Index Entries: Alzheimer’s disease; monoamine oxidase A; polymorphisms; serotonergic pathway; Brazilian population.

Introduction Alzheimer ’s disease (AD), the most common mental disorder in the elderly, leads to cognitive impairment, progressive loss of memory, and other symptoms such as depression and mood alteration. It is characterized by two histopathological hallmarks: neurofibrillary tangles, caused by hyperphosphorylation of the Tau protein, and β-amyloid deposits. Mutations in three genes have been associated with the familial form of AD: presenilin 1, presenilin 2, and the amyloid precursor protein (APP), localized at 14q24.3, 1q31–q42, and 21q21,

respectively. In addition, the ε4 allele of Apolipo protein E (ApoE-ε4) polymorphic locus is the most important associated with late-onset AD (LOAD) sporadic cases. Because depression and mood disorders are observed in patients with AD, different studies have explored whether genes of the serotonergic pathway might be involved with these symptoms (Collier et al., 1996; Lesch et al.; 1996, Li et al., 1997). Serotonin (5-HT) is an important neurotransmitter in the central and peripheral nervous systems, implicated in the control of mood, appetite, sleep, pain, and behavior. Mutations in this gene

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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214 or polymorphisms related to the serotonin pathway, such as the serotonin transporter (5-HTT), could be associated with mood disorders (Wenham et al., 1991; Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Oliveira et al., 1998; Kunugi et al., 2000). The short allele of the 5-HTT gene-linked polymorphic region (5-HTTLPR), involved in the activity of the serotonin transporter gene, has been studied in several mood disorders, including AD, bipolar, and unipolar disease. Some reports suggest that the 5-HT uptake function is reduced in these disorders (Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Kunugi et al., 2000). The search for other polymorphisms associated with AD has been undertaken by many groups of investigators around the world. Several putative candidate loci have been suggested, but most studies have shown controversial results when replicated in different populations, including the 5-HTTLPR polymorphism (Wenham et al., 1991; Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Oliveira et al., 1998; Kunugi et al., 2000). Monoamine oxidase A (MAOA) is a mitochondrial enzyme that catalyzes the degradation of different amines, including neurotransmitters like dopamine, norepinephrine, and serotonin. Several studies have shown that MAOAplays an important role in human behavior and physiology, being associated with mild mental retardation and impulsive aggressive behavior, mood disorders, and alcoholism with antisocial personality (Deckert et al., 1999; Furlong et al., 1999; Kunugi et al., 1999, 2000; Samochowiec et al., 1999; Schuback et al., 1999; Koller et al., 2003; Gutierrez et al., 2004). A functional polymorphism located upstream of this gene was described and consists of a 30-bp repeated sequence and the alleles 2 or 3, which were found to be transcribed 2–10 times more efficiently than others (Sabol et al., 1998). Moreover, the longer alleles (2, 3, and 4) were more frequent in panic disorder patients from German and Italian population than in controls. However, this association was observed only in females, which might be explained because it is an X-linked gene (Deckert et al., 1999). In contrast, no association between any MAOA polymorphic allele and mood disorders was reported by other researchers in the Japanese population, suggesting ethnic differences for association studies with this locus (Kunugi et al., 1999). Taking into account these controversial results, we have undertaken the present study in the Brazil-


Nishimura et al. ian population, aiming to verify (1) whether MAOA polymorphisms are involved in LOAD, and (2) whether the short allele of 5-HTTLPR associated with MAOA polymorphism increases the risk for developing LOAD.

Patients and Methods A total of 128 LOAD patients (46 males and 82 females; mean age, 70.72 ± 8.99) were selected and diagnosed according to NINCDS-ADRDA criteria. To classify the cognitive impairment, neurological and neuropsychological testing, including MiniMental State Exam (MMSE) and Clinical Dementia Rating (CDR), were performed. The age/ethnicmatched control group (45 males and 81 females; mean age, 70.90 ± 9.08) was selected based on the MMSE and/or Blessed Scale, sociocultural and ethnic background. Informed consent was obtained from all subjects included in this study. Genomic DNAwas isolated from peripheral blood according to standard procedures. Analyses of MAOA, 5-HTTLPR, and ApoE polymorphisms were based on previous studies (Wenham et al., 1991; Lesch et al., 1996; Sabol et al., 1998) comparing LOAD patients and healthy controls. The Hardy-Weinberg equilibrium, as well as genotypic and allelic frequencies comparing LOAD patients and controls, was analyzed using χ2 tests, Fisher ’s exact test, and contingency tables. Differences were considered statistically significant at p < 0.05.

Results There were no significant deviations from the Hardy-Weinberg equilibrium for any of the polymorphisms studied. The allele and genotype distribution of the MAOA polymorphism is shown in Table 1. No allele distribution association was observed when the genders were analyzed separately comparing LOAD patients and controls (χ2 = 6.16, df = 3, p = 0.10 for males, and χ2 = 8.69, df = 7, p = 0.27 for females). However, a significant association was observed when both sexes were analyzed together (χ2 = 12.54, df = 4, p = 0.01). Interestingly, allele <1 (2 repeats) and also allele 2 (3.5 repeats) were present only in the LOAD group. For the 5-HTTLPR polymorphism, no association was observed for the allele distribution (p = 0.37), but a borderline association was found for the different genotypes (χ2 = 5.93, df = 2; p = 0.05) (Table 2), and the

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MAOA and AD in Brazilian Population

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Table 1 Allele and Genotype Frequencies of the MAOA Polymorphism in LOAD Patients vs Normal Controls AD MAOAa

Control

Statistical analysis

Genotype

Allele

Genotype

Allele

Male Female (n = 46) (n = 82)

Total (n = 210)

Male Female (n = 45) (n = 81)

Total (n = 207)

<1

0

-

4 (0.02)

0

-

0

1

22 (0.49)

-

84 (0.40)

17

-

71 (0.34)

2 3 4 11 12 13 33 34 44 <11 <13

3 (0.06) 4 (0.02) 21 (0.45) 116 (0.55) 0 2 (0.01) 10 (0.13) 1 (0.01) 39 (0.47) 27 (0.34) 0 1 (0.01) 2 (0.02) 2 (0.02) -

0 26 2 -

a

Genotype

Allele

AD male vs control AD total vs control total: χ2 = 12.54; male: χ2 = 6.16; df = 3; p = 0.10. d f =4; p = 0.01. AD female vs control female: χ2_= 8.69; df = 7; p = 0.27

0 129 (0.63) 7 (0.03) 7 (0.09) 0 40 (0.49) 30 (0.37) 3 (0.04) 1 (0.01) 0 0 -

MAOA alleles: <1 = 2 repeats, 1 = 3 repeats, 2 = 3.5 repeats, 3 = 4 repeats, and 4 = 5 repeats.

Table 2 Genotype and Allele Frequencies of 5HTTLPR in LOAD Patients and Controls LOAD (n = 128) 5-HTTLPR ll ls ss l s

34 (0.26) 72 (0.56) 22 (0.18) 140 (0.55) 116 (0.45)

Controls (n = 126) 48 (0.38) 52 (0.41) 26 (0.21) 148 (0.57) 104 (0.43)

Statistical analysis χ2 = 5.93; df = 2; p = 0.05 p = 0.37, OR = 0.84 (95%) CI = 0.896–1.205

5HTTLPR, 5-hydroxytryptamine transporter-linked polymorphic region. l, long variant; s, short variant.

ApoE-ε4 allele was significantly more frequent in patients than in controls (p < 0.001). The combination of MAOA allele 1 + the short allele of 5-HTTLPR + ApoE-ε4 was observed in 19 patients and in only 4 controls, which was statistically significant (p = 0.001, OR = 0.18 (95%) CI = 0.062–0.570). In contrast, the combination of MAOA allele 3 + the short allele of 5-HTTLPR + ApoE-ε4 did not differ between patients (n = 14) and controls (n = 11) (p = 0.67, OR = 0.77 [95%] CI = 0.339–1.789).


Discussion With the exception of the APOE-ε4 polymorphism, association studies in different populations have shown controversial results, which could be attributed to differences in ethnic background, founder effects, or environmental factors (Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Oliveira et al., 1998; Kunugi et al., 2000; Tsai et al., 2001; Farrer et al., 2003; Luchsinger and Mayeux, 2004).

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216 In the present study, no significant association between the MAOA locus and LOAD was observed when each gender was analyzed separately. However, when both genders were assembled, a statistically significant association was found (p = 0.01). Furthermore, two alleles, allele <1 (2 repeats) and allele 2 (3.5 repeats) were present only in the LOAD group. In addition, a borderline association was found for the genotype distribution of the 5-HTTLPR locus. In a previous study of this locus in another group of Brazilian patients, a strong association between the short allele and LOAD was found (Oliveira et al., 1998). Because the number of patients is relatively small, these results need to be confirmed in independent populations and in a larger number of patients. The ApoE and 5-HTTLPR polymorphisms were studied previously in the Brazilian population. As expected, in the present analysis, the ApoE-ε4 allele was significantly more frequent in patients than in controls (p < 0.001). It has been shown that MAOA alleles 2, 3, and 4 transcribe 2–10 times more efficiently than others, suggesting that they might represent a risk factor for panic disorder in women (Deckert et al., 1999). Carriers of these isoforms showed higher levels of degradation of several neurotransmitters, resulting in decreasing levels of serotonin as well. It is relevant that the short allele of the 5-HTTLPR seems also to decrease the expression of the serotonin transporter, as well as the 5-HT uptake in lymphoblasts. Finally, we analyzed whether there was a preferential segregation of some alleles in patients as compared with controls. Interestingly, the analysis of the MAOA polymorphic locus showed no preferential association of any allele; however, the combination of the MAOA allele 1 + the short allele of 5-HTTLPR + ApoE-ε4 was significantly more frequent in patients than in controls. The analyses of MAOA and serotonin polymorphisms should be replicated in other population studies. The present data reinforce the hypothesis that different genes acting together might play a role in AD susceptibility, although the underlying mechanism is still unknown. Several drugs have been tested in an attempt to retard the progression of this disease, such as tacrine, donepezil, memantine, rivastigmine, and MAOA inhibitors (Youdim and Weinstock, 2004). Recently, a novel neuroprotective drug, TV3326 (N-propargyl-(3R)-aminoindan-5-yl-ethyl, methyl


Nishimura et al. carbamate), which combines the neuroprotective effects of MAOA’s inhibitors and rivastigmine, seems to be useful for AD treatment (Riederer et al., 2004; Youdim and Buccafusco, 2005). It was observed that TV3326 confers neuroprotective activity against cytotoxicity induced by ischemia, reducing neuronal damage (Weinstock et al., 2001; Youdim and Buccafusco, 2005). It has been suggested that this drug acts in APP processing, based on research on rat PC12 and human SH-SY5Y neuroblastoma cells (Youdim and Buccafusco, 2005). It will be of great interest to investigate whether affected AD patients respond differently to potential MAOA inhibitors according to their MAOA genotypes.

Acknowledgments We are extremely grateful for the participation and understanding of the patients and controls. We also thank Dr. Maria Rita Passos-Bueno and her group, Dr. Sergio Matioli, Antonia Maria de Cerqueira, and Constância Urbani for their invaluable help. This work was supported by FAPESPCepid and CNPq.

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217 Samochowiec J., Lesch K. P., Rottmann M., Smolka M., Syagailo Y. V., Okladnova O., et al. (1999) Association of a regulatory polymorphism in the promoter region of the monoamine oxidase A gene with antisocial alcoholism. Psychiatry Res. 86, 67–72. Schuback D. E., Mulligan E. L., Sims K. B., Tivol E. A., Greenberg B. D., Chang S. F., et al. (1999) Screen for MAOA mutations in target human groups. Am. J. Med. Genet. 88, 25–28. Tsai S. J., Hong C. J., Liu T. Y., Cheng C. Y., and Liu H. C. (2001) Association study for a functional serotonin transporter gene polymorphism and late-onset Alzheimer’s disease for Chinese patients. Neuropsychobiology 44, 27–30. Weinstock M., Kirschbaum-Slager N., Lazarovici P., Bejar C., Youdim M. B., and Shoham S. (2001) Neuroprotective effects of novel cholinesterase inhibitors derived from rasagiline as potential anti-Alzheimer drugs. Ann. N. Y. Acad. Sci. 939, 148–161. Wenham P. R., Price W. H., and Blandell G. (1991) Apolipoprotein E genotyping by one-stage PCR. Lancet 337, 1158–1159. Youdim M. B. and Buccafusco J. J. (2005) CNS Targets for multi-functional drugs in the treatment of Alzheimer’s and Parkinson’s diseases. J. Neural Transm. 112, 519–537. Youdim M. B. and Weinstock M. (2004) Therapeutic applications of selective and non-selective inhibitors of monoamine oxidase A and B that do not cause significant tyramine potentiation. Neurotoxicology 25, 243–250.

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