Monoamine oxidases A and B gene polymorphisms in migraine patients

Journal of the Neurological Sciences 228 (2005) 149 – 153 www.elsevier.com/locate/jns

Monoamine oxidases A and B gene polymorphisms in migraine patients Vedrana Filica, Anton Vladicb, Jasminka Stefulja, Lipa Cicin-Saina, Melita Balijac, Zvonimir Sucicb, Branimir Jerneja,* a

Laboratory of Neurochemistry and Molecular Neurobiology, Dept. of Molecular Biology, Rudjer Boskovic Institute, Bijenicka 54, Zagreb HR-10000, Croatia b Sveti Duh General Hospital, Department of Neurology and Department of Radiology, Sveti Duh 64, Zagreb, Croatia c Croatian Institute of Transfusion Medicine, Petrova 3, Zagreb, Croatia Received 2 June 2004; received in revised form 26 August 2004; accepted 12 November 2004 Available online 24 December 2004

Abstract Abnormal cortical activity and brainstem functioning are considered the possible etiopathogenetic factors of migraine. Monoamine oxidase A and B (MAO-A and -B) regulate the levels of monoamine neurotransmitters, so changes in their activity could participate in migraine pathogenesis. We have investigated the possible association of MAO-A and -B alleles and haplotypes with two common types of migraine, i.e. migraine without aura (MO) and migraine with aura (MA), on the sample of 110 migraineours (80 MO and 30 MA) and 150 controls. MAO-A promoter and MAO-B intron 13 polymorphisms were genotyped by the PCR-based methods. In addition, we have reevaluated the reported association between MAO-B intron 13 polymorphism and platelet MAO-B activity. The platelet MAO-B activity was determined fluorimetrically using kynuramine as a substrate. We have found a tendency toward association of the shorter variant of MAO-A gene promoter with migraine without aura in male subjects. Regarding investigated MAO-B polymorphism, no association with migraine or with platelet MAO-B activity was found. The suggestive association of the variant in MAO-A gene with migraine is considered worthy of independent replication. On the other hand, further studies on MAO-B polymorphism in migraine do not seem promising. D 2004 Elsevier B.V. All rights reserved. Keywords: Migraine; Monoamine oxidase; Polymorphism; Serotonin; Noradrenaline; Platelet

1. Introduction Migraine is a neurovascular disease that affects about 15% of the western population [1,2]. Epidemiological studies point that genetic factors play an important role in the etiology of this complex disorder [3]. However, until now, the causative gene has been identified only for a rare familial hemiplegic migraine [4], while for more common types of migraine, i.e. migraine without aura (MO) and migraine with aura (MA), a number of association studies still have not yielded fruitful results [5,6]. One way by which genetic factors could affect the migraine etiopathogenesis is by the control of the metabolism of monoaminergic neurotransmitters. Namely, according to

* Corresponding author. Tel.: +385 1 4561 150; fax: +385 1 4680 228. E-mail address: [email protected] (B. Jernej). 0022-510X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2004.11.045

the current theories, the primary causes of migraine pain could be abnormal cortical activity and brainstem nuclei dysfunction [7], with foci in dorsal raphe nuclei and locus coeruleus [8]. Sensory cortices are under the modulation of noradrenergic, cholinergic and serotonergic inputs from brainstem nuclei [9], so it is possible that altered neurotransmitter, especially serotonin and noradrenaline, levels, play a certain role in the migraine etiopathogenesis. Isoenzymes monoamine oxidases A and B (MAO-A and -B) catalyze oxidative deamination, an essential step in the catabolism of monoamine neurotransmitters and thus participate in functional regulation [10,11]. Genes encoding human MAO-A and -B are located on the short arm of the chromosome X [12,13]. They are arranged in tail-totail configuration [14] and both contain common polymorphisms. Upstream variable number of tandem repeats polymorphism in MAO-A gene (MAOA-uVNTR) is located in the promoter region and contains a 30 bp long

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and 50 men, without personal or family history of headache or other neurological or psychiatric disorders, as reported in a written interview. The correlation between MAO-B gene polymorphism and platelet MAO-B activity was studied on a separate group of 70 healthy male blood donors, nonsmokers, age 32.4F9.4 years. All participants signed an informed consent before entering the study.

repeated sequence [15]. Several allelic variants (with 2, 3, 3.5, 4, 4.5, 5 and 6 repeats) have been identified, although variants with 3 and 4 repeats constituted more than 97% of the alleles in all reported control samples [15–19]. Expression studies pointed to the functional relevance of MAOA-uVNTR polymorphism by showing that 4-repeats allele was linked to more efficient transcription than 3-repeats allele [15,20]; this relation, however, could not be demonstrated in the postmortem samples of the human brain [21]. MAO-B gene contains A/G dimorphism in intron 13, located 36 bp upstream from the intron 13–exon 14 boundary [22,23]. Initial study of Garpenstrand et al. [24] reported that male individuals with A-allele show significantly lower MAO-B activity in platelets than individuals with G-allele, while the subsequent study on the brain MAO-B activity reported opposite effects of this polymorphism [21]. Association of MAO-A and -B polymorphisms with various neuropsychiatric disorders has been investigated, with results being inconclusive so far [25,26]. Our previous work on the migraine was focused, by the use of platelet model, on the MAO activity [27]. The present study investigates the association of migraine with polymorphisms in MAO-A and -B genes. In addition, it also reevaluates, on a larger sample and different ethnicity, the reported correlation between MAO-B polymorphism and the platelet MAO activity [24].

2.2. Genotyping Genomic DNA was isolated from the peripheral blood using standard phenol chloroform extraction. For MAOAuVNTR polymorphism 100 ng DNA was amplified by polymerase chain reaction (PCR) in a mixture containing 0.2 AM primers, 200 AM each dNTP, 1.5 mM MgCl2 and 0.01 U AL 1 Taq DNA polymerase (Promega) in a final volume of 20 AL. The primer sequences were taken from the previous study [15]. Cycle conditions were as follows: 2 min at 95 8C, 35 cycles (30 s at 95 8C, 30 s at 61 8C and 40 s at 72 8C), and 7 min at 72 8C. Genotyping was performed according to the length of PCR products (276, 306, 321, 336 and 351 bp for 2, 3, 3.5, 4 and 4.5 repeats, respectively) separated on the 10% polyacrylamide gel. MAO-B dimorphism was genotyped by allele-specific oligonucleotide PCR (ASO-PCR), using published primer sequences [22]. The reaction mixture of 15 AL contained 75 ng of genomic DNA, 0.2 AM primers, 50 AM each dNTP, 1.5 mM MgCl2 and 0.003 U AL 1 Ampli Taq DNA Gold polymerase (Perkin Elmer). The cycling conditions were as follows: 95 8C for 10 min (hot start), 35 cycles (30 s at 95 8C, 30 s at 61 8C and 40 s at 72 8C), and 72 8C for 5 min. The presence or the absence of 663 bp long PCR product was checked after electrophoresis on 1.6% agarose gel stained with ethidium bromide.

2. Materials and methods 2.1. Subjects Blood samples were collected from the clinic outpatients of Department of Neurology of Sveti Duh General Hospital, Zagreb. The study included 110 patients suffering from migraine (age 35.7F14.0 years): 80 from migraine without aura (56 women and 24 men) and 30 from migraine with aura (24 women and 6 men). The diagnoses were made according to the International Headache Society criteria [28]. The study was approved by the Ethics committee of the Medical faculty, University of Zagreb. The control group consisted of 150 healthy blood donors (age 41.4F11.2 years), 100 women

2.3. Measurement of platelet MAO-B activity Preparation of platelet-rich plasma (PRP) was done by the method described previously [27]. Enzyme velocity was expressed as nanomoles 4-HQ per 108 platelets per 60 min and K M as AM concentration of kynuramine.

Table 1 Allele and genotype frequencies of MAOA-uVNTR polymorphism among migraine patients and controls Sample (N)

Allele, N (%) 4

Females

Males

controls (96) MO (55) MA (22) controls (49) MO (23) MA (6)

127 67 33 34 10 3

p 3

(66.1) (60.9) (75.0) (69.4) (43.5) (50.0)

MO=migraine without aura; MA=migraine with aura.

65 43 11 15 13 3

(33.9) (39.1) (25.0) (30.6) (56.5) (50.0)

0.3840 0.2878 0.0423*

Genotype, N (%)

p

4/4

4/3

3/3

46 (47.9) 18 (32.7) 12 (54.6)

35 (36.5) 31 (56.4) 9 (40.9)

15 (15.6) 6 (10.9) 1 (4.5)

0.0598 0.3915

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151

Table 2 Allele and genotype frequencies of A/G dimorphism among migraine patients and controls Sample (N)

Allele, N (%) A

Females

Males

controls (100) MO (56) MA (24) controls (50) MO (24) MA (6)

110 58 29 23 14 3

p

Genotype, N (%) A/A

A/G

G/G

0.6363 0.5216

37 (37.0) 16 (28.6) 11 (45.8)

36 (36.0) 26 (46.4) 7 (29.2)

27 (27.0) 14 (25.0) 6 (25.0)

G (55.0) (51.8) (60.4) (46.0) (58.3) (50.0)

90 54 19 27 10 3

(45.0) (48.2) (39.6) (54.0) (41.7) (50.0)

p

0.4091 0.7118

0.4567

MO=migraine without aura; MA=migraine with aura.

2.4. Statistical analysis Statistical analyses were carried out using InStat (Version 3.01 for Windows), GraphPad Software, Inc., USA. Due to the X-chromosomal location of the investigated genes, males and females were considered separately. Differences in genotype distributions between case and control groups, as well as the presence of Hardy–Weinberg equilibrium, were tested by the v 2 test for independence. Comparisons of allele frequencies were performed by the two-sided Fisher’s Exact Test (FET). Haplotype frequencies in females were estimated from genotypic data by maximum-likelihood method based on the expectation maximization algorithm, using the Arlequin program (version 2.000). Comparison of the groups for the estimated frequencies of haplotypes was performed using the log-likelihood ratio test. Correction for multiple testing was carried using the Bonferroni procedure (the level of significance was set at a=0.017). Testing for normality of kinetic parameters (V max, K M and V max/K M) of MAO-B was performed using one-sample t-test. Mann– Whitney test was used to evaluate the differences in MAOB-activity means between individuals grouped according to the intron 13 genotype.

3. Results 3.1. MAO-A and -B gene polymorphisms in migraine In our samples, we have detected five allelic variants of the MAO-A promoter: with 2, 3, 3.5, 4 and 4.5 repeats. Since alleles with 4.5, 3.5 and 2 repeats constituted only 2% of all alleles, individuals carrying those alleles were excluded from statistical analyses. Genotype frequencies in females accorded with Hardy–Weinberg equilibrium for both MAO-A (controls: v 2=1.618, df=2, p=0.4454; MO: v 2 =0.9551, df=2, p=0.6203; MA: v 2 =0.3922, df=2, p=0.8219) and MAO-B (controls: v 2 =4.053, df=2, p =0.1318; MO: v 2 =0.1434, df=2, p =0.9308; MA: v 2=1.489, df=2, p=0.4750) polymorphism. Genotype and allele frequencies of the MAO-A promoter polymorphism did not differ between female controls and patients, regardless of the migraine type (Table 1). On the other hand, male patients with migraine without aura

showed marginally increased ( p=0.0423, Table 1) frequency of the allele with 3 repeats, as compared to controls. However, the statistical significance of this finding was lost after correction for multiple testing. Genotype and allele frequencies of the MAO-B dimorphism did not differ between female controls and patients (Table 2). Similarly, male controls and patients did not show differences in allele frequencies in any of the samples (Table 2). The frequencies of the corresponding MAO-A/MAO-B haplotypes (Table 3) also did not differ significantly between control and patient population (females: log-likelihood ratio test, MO: G =3.746, df =3, p =0.2978; MA: G =1.182, df =3, p=0.7673; MO males: v 2=4.803, df=3, p=0.1868). 3.2. The correlation of MAO-B polymorphism with platelet MAO activity On the sample of 70 healthy nonsmoking males, maximal enzyme velocity (V max) ranged from 5.93 to 23.81 nmol per 108 platelets per 60 min (the mean value 11.37F4.18 nmol per 108 platelets per 60 min), Michaelis constant (K M) ranged from 3.25 to 8.24 AM (the mean value 5.06F1.05 AM), and the kinetic product (V max/K M) ranged from 1.14 to 4.61 (the mean value 2.32F0.94). Distributions of enzyme velocity and efficacy failed at normality testing ( pb0.05), while only K M values gave normal distribution (one-sample t-test; KS=0.14; pN0.05). 42 (60%) of individuals carried A-allele, while 28 (40%) had G-allele. There were no statistically significant differences between these Table 3 MAO-A/MAO-B haplotype frequencies in control subjects and in two different types of migraine MAO-A/MAO-B haplotype

Males, N (%)

Females, N (%)

Control (n=49)

MO (n=23)

Control (n=96)

MO (n=55)

MA (n=22)

4/A

13 (26.5) 21 (42.9) 10 (20.4) 5 (10.2)

5 (21.7) 5 (21.7) 9 (39.1) 4 (17.4)

34 (34.9) 30 (31.2) 18 (19.2) 14 (14.6)

21 (38.0) 13 (22.9) 7 (12.9) 14 (26.2)

10 (43.3) 7 (31.6) 3 (13.5) 2 (11.5)

4/G 3/A 3/G

MO=migraine without aura; MA=migraine with aura.

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two groups in the mean values of V max ( p=0.7102) or V max/ K M ( p=0.7326).

4. Discussion Biochemical studies of MAO-B in migraine, including our recent study [27], mostly failed to show a correlation between platelet MAO activity and this disorder [29]. The reason could be the different regulation of the enzyme activity in the blood cells vs. the brain [30], supported also by the lack of intraindividual correlation between platelet and brain MAO-B activity [31,32]. In the present work, we used an alternative approach to study the involvement of the mentioned enzymes in the migraine ethiopathogenesis, by investigating the potential association of enzyme’s gene variants with the disorder. In MAO-A gene we focused on the promoter polymorphism that was demonstrated to have functional consequences in different cell lines [15,20]. From 250 alleles of the control sample, alleles with 3 and 4 repeats amounted to 98%, which is in accordance with literature reports [18,19,33]. We have found a weak association between the shorter allele and MO in males ( p=0.0423), with the indication of the same direction in women ( p=0.0598). The significance of the association obtained on males was, however, lost after Bonferroni correction. Nevertheless, taking into acount potential functionality of the investigated polymorphism, as well as the pioneering character of the study, we would not like to discard this observation in order to avoid false negative results (type II error). In a far projection, the reduction of MAO-A activity in migraine would fit well into hypothesis of monoaminergic hyperactivity observed in the brain stem nuclei of migraineours [8]. In MAO-B gene we were focused on the A/G dimorphism in intron 13, which was reported to have the effect on the enzyme activity in platelets [24] and in brain [21]. We have found no association between this polymorphism and migraine ( p values ranged from 0.4 to 0.7, Table 2). Balciuniene et al. [21] have found that particular combination of MAOA-uVNTR and MAO-B A/G polymorphisms (resulting with haplotype 3/G) is linked to lower MAO-A enzyme levels in human brain. Haplotypic analyses in our study, however, did not demonstrate association of the suspected haplotype with the migraine. The reported opposite effect of the A/G polymorphism in MAO-B gene on the enzyme activity in blood platelets [24] and in brain [21] prompted us to reevaluate its functional consequences on the platelet MAO-B activity. As there are multiple influences upon platelet MAO activity, a special attention was given to obtain a homogenous sample of healthy male subjects (N=70), younger than 50 years and nonsmokers. No correlation was found between platelet MAO activity and polymorphism in its gene. This result, in contrast to the above mentioned study of Garpenstrand et al. [24], point to the

absence of functional relevance of A/G dimorphism, at least in platelets. The possible explanation for the contradictory results could be related to the ethnical influences. However, having in mind that platelet MAO activity is influenced by variety of factors (age, sex, alcohol and tobacco use, personality characteristics, etc.; for review, see Ref. [34]), it seems more likely that the platelet model is not valid for this purpose. Our results, however, do not exclude the possibility of functional effect of this polymorphism on the MAO-B activity in the brain. In conclusion, our results speak against further studies on the association of either MO or MA with MAO-B A/G polymorphism. On the other hand, the findings on MAOAuVNTR polymorphism in MO seems indicative, advocating the independent replication.

Acknowledgement This study was supported by Croatian Ministry of Science and Technology (grant number 0980002).

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