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A promoter polymorphism in the monoamine oxidase A gene and its relationships to monoamine metabolite concentrations in CSF of healthy volunteers
Journal of Psychiatric Research 34 (2000) 239±244
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A promoter polymorphism in the monoamine oxidase A gene and its relationships to monoamine metabolite concentrations in p CSF of healthy volunteers Erik G. JoÈnsson a,*, Nadine Norton b, J. Petter Gustavsson c, Lars Oreland d, Michael J. Owen b, GoÈran C. Sedvall a a
Department of Clinical Neuroscience, Psychiatry Section, Karolinska Institute, SE-171 76 Stockholm, Sweden b Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardi, UK c Department of Health Sciences, University of SkoÈvde, SkoÈvde, Sweden d Department of Neuroscience, Section of Pharmacology, University of Uppsala, Uppsala, Sweden Received 4 November 1999; received in revised form 15 February 2000; accepted 23 March 2000
Abstract Concentrations of monoamine metabolites (MM) in lumbar cerebrospinal ¯uid (CSF) have been used extensively as indirect estimates of monoamine turnover in the brain. We investigated possible relationships between a putative functional promoter polymorphism in the monoamine oxidase A (MAOA) gene and CSF concentrations of homovanillic acid (HVA), 5hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in healthy volunteers (n = 88). Among women (n = 37), those carrying at least one copy of the alleles associated with more ecient transcription displayed higher concentrations of HVA ( p = 0.01) and 5-HIAA ( p = 0.01). In men (n = 51), however, there was a tendency in the opposite direction. The results suggest that MAOA genotypes may participate dierentially in the regulation of dopamine and serotonin turnover rates under presumed steady state in the central nervous system. The results should be interpreted with caution until replicated because of the limited sample size. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Monoamine oxidase A gene; Monoamine metabolites (HVA; 5-HIAA; MHPG); Cerebrospinal ¯uid
1. Introduction Concentrations of the major monoamine metabolites (MM) 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in lumbar cerebrospinal ¯uid (CSF) have been used extensively as indirect measures of monoamine metabolism and turnover in the brain of humans. Studies of human twins indicate that CSF 5Part of this investigation has been presented at the 7th World Congress on Psychiatric Genetics, Monterrey, California, October 14±18, 1999. * Corresponding author. Tel.: +46-8-51772626; fax: +46-8346563. E-mail address: [email protected] (E.G. JoÈnsson). p
HIAA and HVA levels are under familial in¯uence of both genetic and environmental origin, while MHPG is under major genetic in¯uence (Oxenstierna et al., 1986). In rhesus monkeys signi®cant portions of CSF 5-HIAA, the major serotonin metabolite, HVA, the major dopamine metabolite, and MHPG, the major norepinephrine metabolite in the central nervous system, have been shown to be determined by genetic mechanisms (Higley et al., 1993). Monoamine oxidase (E.C. 1.4.3.4) is a mitochondrial enzyme involved in the degradation of biogenic amines including dopamine, serotonin, and norepinephrine. In humans two isozymes exist: monoamine oxidase A (MAOA), which preferentially deaminates serotonin and norepinephrine, and monoamine oxidase B (MAOB) catalyzing degradation of phenylethylamines and benzylamine
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(Bach et al., 1988; Cawthon et al., 1981; Donnelly and Murphy, 1977). With regard to dopamine, at least in man, both isoenzymes participate with some preponderance of MAOB (StenstroÈm et al., 1987). This makes both the MAO genes, located on chromosome Xp11.23±Xp11.4 (Kochersperger et al., 1986; Lan et al., 1989; Levy et al., 1989; Ozelius et al., 1988; Pintar et al., 1981), of interest in studies of mechanisms for regulation of monoamine turnover and metabolism. Recently a MAOA promoter variable number tandem repeat (VNTR) polymorphism has been reported (Deckert et al., 1999; Sabol et al., 1998). This VNTR consists of exact repeats of a 30-bp sequence. Two to ®ve exact repeats have been reported. Also, the four complete repeats are followed by a half repeat consisting of the ®rst 15 bp of the repeated motif, giving the following repeat nr: 2, 3, 3.5, 4, and 5. In transfection experiments both Deckert et al.(1999) and Sabol et al.(1998) have shown that alleles with 3.5 and 4 copies are transcribed more eciently than those with 3 copies. In the present study we have examined this MAOA VNTR polymorphism for possible relationships to concentrations of 5-HIAA, HVA, and MHPG in lumbar CSF from healthy Swedish volunteers. 2. Methods 2.1. Healthy human volunteers The investigation was carried out in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of the Karolinska Hospital, Stockholm. Informed consent of the subjects was obtained after the nature of the procedures had been fully explained. The characteristics and assessment of the majority of subjects (n = 66) participating in the present study have previously been described (JoÈnsson et al., 1996). In this study another 22 Caucasian individuals were added. Of these subjects 16 were initially drawn from a population register from Stockholm county council to take part in a study regarding CSF circulation including lumbar puncture (Oxenstierna et al., 1996). Otherwise these subjects were assessed in a similar way as the original 66 individuals, who were recruited predominantly among students or hospital sta. Lumbar puncture (LP) was performed in all subjects. Height was also recorded. Back length, de®ned as the distance between the external occipital protuberance and the insertion point of the lumbar needle with the subject in the lying position, was measured in 63 subjects. Eight to 19 years later a structured interview was performed by a psychiatrist (EJ) to assess psychiatric morbidity (DSM-III-R; American Psychiatric Association, 1987), somatic illness and presence of mental and nervous
system disorders among relatives. Subjects completed a questionnaire regarding smoking habits. Hospital records were obtained and examined for diagnosis. Genealogical data for antecedents up to the third degree were obtained from parish registers to assess the origin of the individuals. Subjects who reported any life time psychiatric disorder were excluded. Of the subjects 51 were men and 37 women. The age range at the time of the present study was 29±56 years, with a mean of 41 years. The mean age at LP was 27 years, age range 19±43. Thirty six were university graduates. Twenty one subjects had a family history of major mental illness de®ned as at least one ®rst or second degree relative with schizophrenia, schizoaective disorder, bipolar disorder, recurrent unipolar disorder, other non-organic psychosis, or who had committed suicide. Of the subjects 53 were or had been regular tobacco users, 18 were non smokers or had only used tobacco once or a few times in their life, while data were missing for 11 individuals. Of the women, 15 used oral contraceptives at LP, 20 did not, while data were missing for two individuals. Except for oral contraceptives all participants were drug free at LP. Genealogical data implicated that 89 and 5% of the genes originated in ancestors born in Sweden and Finland, respectively, and the remaining 6% were distributed over eight European countries. 2.2. CSF MM concentrations All subjects had at least 8 h of bed rest in the hospital, abstaining from food and smoking. CSF samples were obtained by LP between 8 and 9 a.m.with the subjects in the sitting (n = 41) or recumbent (n = 47) position. Samples of 12.5 ml CSF were drawn according to a standardized sampling procedure (Sedvall et al., 1980). Samples were stored at below ÿ208C and analyzed within 2 months. HVA, 5-HIAA, and MHPG concentrations were measured by mass fragmentography with deuterium labelled internal standards (Swahn et al., 1976). 2.3. Genotype analyses Venous blood was taken from all individuals into EDTA-containing tubes. DNA was isolated as previously described (Geijer et al., 1994). The 186±276 bp fragments containing the MAOA VNTR were ampli®ed using the following primers: forward ¯uorescently labelled 5'-GCC CAG GCT GCT CCA GAA A-3 ' and reverse 5 '-GAA CGG ACG CTC CAT TCG GA-3'. The PCR conditions were as follows: 200 mM dNTPs, 1 Taq Gold PCR buer II (Perkin Elmer), 2.5 mM Mg, 1 unit Taq Gold (Perkin Elmer), and 48 ng DNA, with an initial denaturating
E.G. JoÈnsson et al. / Journal of Psychiatric Research 34 (2000) 239±244
period at 958C for 10 min, followed by 35 cycles of 95 (45 sec), 62 (45 sec), and 628C (10 min). The PCR products were electrophoresed on 10% denaturing polyacrylamide gels on an ABI 373 DNA sequencer, and analysed using the ABI GENESCAN and GENOTYPER software. 2.4. Data analysis Subjects were grouped into those with or without 3.5/4 alleles, respectively, because the MAOA alleles containing 3.5 and 4 alleles have been shown to induce a more eective transcription than other alleles (Sabol et al., 1998). In a previous study, of the majority of the present sample, back length was found to be a signi®cant confounding variable for MM concentrations in CSF (JoÈnsson et al., 1996). To make it possible to correct MM for back length, back length values, based on the relationship between back length and height (r = 0.73, p < 0.0001), were constructed for those where only height had been measured (n = 26). Analysis of covariance (ANCOVA) was used to adjust for dierences in CSF levels due to back length. Because correcting MM concentrations for back length is still controversial (JoÈnsson et al., 1997), analyses were also performed with MM data uncorrected for back length. Analysis of variance (ANOVA) was used. Signi®cance level was de®ned as a p-value less than 0.05. Because of the exploratory nature of the investigation, no corrections were made for multiple comparisons. However, the reader is reminded of the increased potential for type I errors due to the large number of analyses performed (Grove and Andreasen, 1982). 3. Results The results shown in Table 1 demonstrate the stat-
241
istical data analysis performed on MM residuals corrected and uncorrected for back length. MM means and standard deviations (SD) are presented as uncorrected values. Four dierent alleles were detected in the present sample. In one women an allele with only two repeats was found. The allele frequencies were 0.01 (2 repeats), 0.31 (3 repeats), 0.02 (3.5 repeats), and 0.66 (4 repeats), respectively. Among men the allele frequencies were 0.22 (3 repeats), 0.02 (3.5 repeats), and 0.77 (4 repeats), respectively. In women the allele frequencies were 0.01 (2 repeats), 0.38 (3 repeats), 0.03 (3.5 repeats), and 0.58 (4 repeats), respectively, distributed on the following genotypes: 2/3 (3%), 3/3 (11%), 3/3.5 (3%), 3/4 (49%), 3.5/4 (3%), and 4/4 (32%). As the MAOA gene is located on the X chromosome, each gender was analysed separately. In women a relationship was indicated between the MAOA polymorphism and HVA-levels (F = 5.22, d.f.=1, p = 0.029 and F = 6.65, p = 0.015 uncorrected and corrected for back length, respectively). Women with genotypes including the 3.5 or 4 repeat alleles had higher HVA concentrations than women without these alleles. A similar relationship was obtained when the female subjects were analyzed for MAOA genotypes with regard to 5-HIAA levels. Women with genotypes including 3.5 or 4 repeat alleles displayed higher 5HIAA levels than those without these alleles (F = 7.47, d.f.=1, p = 0.010 and F = 7.31, p = 0.011 uncorrected and corrected for back length, respectively). However, no relationship was found in women between the MAOA polymorphism and MHPG levels. Women were also grouped into subjects only carrying alleles suggested to give rise to high transcription (genotypes 3.5/4 and 4/4), low transcription (2/3, 3/3), or subjects carrying both these types of alleles (3/3.5, 3/ 4). When compared vs CSF MMs the following results
Table 1 Comparisons (ANOVA) between monoamine oxidase A (MAOA) genotypes and homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) concentrations in lumbar cerebrospinal ¯uid of healthy volunteers HVA
5-HIAA
MHPG
Sex
Allele
n
Mean2SD (nmol/L)
Fa pa
Fb pb
Mean2SD (nmol/L)
Fa pa
Fb pb
Mean2SD (nmol/L)
Fa pa
Fb pb
Men
3.5/4 Other Genotype 3.5/4c Other 3.5/4+4/4 3/3.5+3/4 2/3+3/3
40 11
154261 208290
F = 3.43 p = 0.070
F = 5.33 p = 0.025
89235 108240
F = 1.04 p = 0.313
F = 2.47 p = 0.122
4227 4629
F = 0.85 p = 0.361
F = 1.54 p = 0.221
32 5 13 19 5
209272 133245 192253 221282 133245
F = 6.65 p = 0.015 F = 3.75 p = 0.034
F = 5.22 p = 0.029 F = 3.32 p = 0.048
113235 68215 106233 117237 68215
F = 7.31 p = 0.011 F = 4.14 p = 0.025
F = 7.47 p = 0.010 F = 4.21 p = 0.023
4226 3722 4126 4226 3722
F = 2.57 p = 0.118 F = 1.42 p = 0.256
F = 2.58 p = 0.117 F = 1.46 p = 0.246
Women Women
a
Statistical comparisons corrected for back length. Statistical comparisons uncorrected for back length. c Genotypes containing alleles 3.5 and/or 4.
b
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emerged: CSF HVA (F = 3.32, d.f.=2, p = 0.048 and F = 3.75, p = 0.034 uncorrected and corrected for backlength, respectively), CSF 5-HIAA (F = 4.21, d.f.=2, p = 0.023 and F = 4.14, p = 0.025 uncorrected and corrected for backlength, respectively), and MHPG (F = 1.46, d.f.=2, p = 0.246 and F = 1.42, p=0.256 uncorrected and corrected for backlength, respectively). Without correction for back length there was a relationship in men (F = 5.33, d.f.=1, p = 0.025) with lower CSF HVA-levels in subjects with the 3.5 or 4 repeat alleles as compared to those lacking these alleles. However, after correction for back length this relationship was reduced to a trend (F = 3.43, p = 0.070). No relationships was found in men between the MAOA polymorphism and CSF 5-HIAA and MHPG concentrations, respectively. 4. Discussion To our knowledge the present publication is the ®rst comparing MAOA genotypes and lumbar CSF MM in healthy human subjects. In women, relationships were found between MAOA genotypes and CSF concentrations of HVA and 5-HIAA. Women with genotypes containing alleles with 3.5 and/or 4 repeats, reported to induce a more eective monoamine oxidase transcription (Deckert et al., 1999; Sabol et al., 1998), were found to have higher levels of HVA and 5-HIAA. A similar tendency was also found with regard to MHPG in women, although this dierence was not signi®cant. It would seem reasonable that a higher amount of a converting enzyme may contribute to higher levels of a resulting degradation product and, furthermore, treatment with an MAOA inhibitor results in a decrease in CSF levels of monoamine transmitter metabolites (Major et al., 1979). The experiments by Sabol et al. (1998) showed that the MAOA activities diered considerably when the dierent MAOA gene promoter VNTR alleles were transfected to cell lines. Post mortem studies on human brain tissues, on the other hand, indicate that variation in MAOA activity is remarkably low (Fowler et al., 1980). Thus, it remains to be shown that the present MAOA gene polymorphism is associated with variation in enzyme activity in vivo. In men no signi®cant relationships emerged, although a trend in the opposite direction as compared to women was found between MAOA alleles and HVA levels. This may indicate that there are marked dierences between men and women with regard to the strength of these relationships. There have been reports of gender dierences with regard to CSF monoamine metabolites (Blennow et al., 1993; Constantino and Murphy, 1996; Nordin et al., 1995). If there is a real dierence between men and
women with regard to the relationships between MAOA genotypes and monoamine metabolite levels this may indicate certain confounding factors which dier between the sexes. In¯uence of steroids has been discussed as one such potential confounder (Best et al., 1992; JoÈnsson et al., 1997). Interstingly, in the present sample female users of contraceptives had signi®cantly lower CSF-levels of 5-HIAA (t = 2.40, d.f.=33, p = 0.023) and MHPG (t = 2.25, d.f.=33, p = 0.031) than non-users. These dierences were not signi®cantly in¯uenced by back length (data not shown). Similar dierences with regard to CSF MHPG, but not 5HIAA levels have previously been reported (Eklundh et al., 1994; TraÈskman-Bendz, 1980). However, we did not ®nd any MAOA genotype dierence between users and non-users of contraceptives (Fisher's exact test p = 0.141) indicating that the relationships between MAOA genotypes and monoamine metabolite levels are not signi®cantly in¯uenced by the use of contraceptives. Tobacco use has been suggested to signi®cantly in¯uence CSF HVA (Geracioti Jr et al., 1999) and also to considerably inhibit the activity of monoamine oxidase (Fowler et al., 1996a,b). In the present sample, however, no signi®cant dierences were found when use or non-use of tobacco were compared vs MAOA allele/genotype constalletions or levels of HVA, 5HIAA, or MHPG in each sex separately or with the two sexes combined, respectively (data not shown). This result, together with the previously reported small variation in enzyme activity in post mortem brain tissue (Fowler et al., 1980), may indicate that the associations found are not the result of a direct cause relationship. An alternative explanation would be that the in¯uence of the dierent MAOA alleles on the expression of enzyme activity during prenatal development will induce life-long eects on brain monoamine systems, similar to those induced in rats by prenatal MAOA inhibition (Whitaker-Azmitia et al., 1994). The signi®cant CSF HVA and 5-HIAA dierences with regard to MAOA genotypes in the present study may be chance ®ndings. The sample size is limited. Also considering the many variables tested, type I errors can be expected to emerge. There were six primarily independent investigations performed (genotypes of one polymorphism vs concentrations of three monoamine metabolites in two sexes) in the total sample. This gives a p-value of less than 0.008 to be considered signi®cant using Bonferroni's correction. Thus, applying this correction in the total sample no relationship would remain signi®cant. This means that until the present results are replicated in independent investigations, they must be regarded as preliminary and should be interpreted with caution. Dopamine, serotonin and norepinephrine transmission have been implicated in aective disorder and
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schizophrenia (Potter and Manji, 1994; van Kammen and Antelman, 1984; van Rossum, 1967; Woolley and Shaw, 1954). The present tentative ®ndings indicate that MAOA genotypes participate dierentially in the regulation of dopaminergic and serotonergic transmission in the central nervous system. Therefore, the variables recorded can be regarded as valid parameters to examine for changes in these psychiatric disorders. The present results do not favour a dierential in¯uence of MAOA gene activities on MHPG concentrations in lumbar CSF. Acknowledgements This study was supported by grants from the Swedish Medical Research Council (03560), the National Institute of Mental Health (44814), the Medical Research Council (UK), and the SoÈderstroÈm-KoÈnigska Foundation. We thank Kjerstin Lind and Alexandra Tylec, Department of Clinical Neuroscience, Karolinska Hospital for technical assistance. References American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 3rd ed. Washington DC: American Psychiatric Association, 1987 revised. Bach AW, Lan NC, Johnson DL, Abell CW, Bembenek ME, Kwan SW, Seeburg PH, Shih JC. cDNA cloning of human liver monoamine oxidase A and B: molecular basis of dierences in enzymatic properties. Proceedings of the National Academy of Sciences of the United States of America 1988;85:4934±8. Best NR, Rees MP, Barlow DH, Cowen PJ. Eect of estradiol implant on noradrenergic function and mood in menopausal subjects. Psychoneuroendocrinology 1992;17:87±93. Blennow K, Wallin A, Gottfries CG, Karlsson I, MaÊnsson JE, Skoog I, WikkelsoÈ C, Svennerholm L. Cerebrospinal ¯uid monoamine metabolites in 114 healthy individuals. Eur Neuropsychopharmacology 1993;3:55±61. Cawthon RM, Printar JE, Haseltine FP, Breake®eld XO. Dierences in the structure of A and B forms of human monoamine oxidase. Journal of Neurochemistry 1981;37:363±72. Constantino JN, Murphy DL. Monoamine metabolites in `leftover' newborn human cerebrospinal ¯uid. A potential resource for biobehavioral research. Psychiatry Research 1996;65:129±42. Deckert J, Catalano M, Syagailo YV, Bosi M, Okladnova O, Di Bella D, NoÈthen MM, Maei P, Franke P, Fritze J, Maier W, Propping P, Beckmann H, Bellodi L, Lesch KP. Excess of high activity monoamine oxidase A gene promoter alleles in female patients with panic disorder. Human Molecular Genetics 1999;8:621±4. Donnelly CH, Murphy DL. Substrate and inhibitor-related characteristics of human platelet monoamine oxidase. Biochemical Pharmacology 1977;26:853±8. Eklundh T, FernstroÈm V, Nordin C. In¯uence of tapping time and atmospheric pressure on concentrations of monoamine metabolites in the cerebrospinal ¯uid: a prospective study in female volunteers. Journal of Psychiatric Research 1994;28:511±7. Fowler CJ, Wiberg AÊ, Oreland L, Marcusson J, Winblad B. The eect of age on the activity and molecular properties of human
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A promoter polymorphism in the monoamine oxidase A gene and its relationships to monoamine metabolite concentrations in p CSF of healthy volunteers Erik G. JoÈnsson a,*, Nadine Norton b, J. Petter Gustavsson c, Lars Oreland d, Michael J. Owen b, GoÈran C. Sedvall a a
Department of Clinical Neuroscience, Psychiatry Section, Karolinska Institute, SE-171 76 Stockholm, Sweden b Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardi, UK c Department of Health Sciences, University of SkoÈvde, SkoÈvde, Sweden d Department of Neuroscience, Section of Pharmacology, University of Uppsala, Uppsala, Sweden Received 4 November 1999; received in revised form 15 February 2000; accepted 23 March 2000
Abstract Concentrations of monoamine metabolites (MM) in lumbar cerebrospinal ¯uid (CSF) have been used extensively as indirect estimates of monoamine turnover in the brain. We investigated possible relationships between a putative functional promoter polymorphism in the monoamine oxidase A (MAOA) gene and CSF concentrations of homovanillic acid (HVA), 5hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in healthy volunteers (n = 88). Among women (n = 37), those carrying at least one copy of the alleles associated with more ecient transcription displayed higher concentrations of HVA ( p = 0.01) and 5-HIAA ( p = 0.01). In men (n = 51), however, there was a tendency in the opposite direction. The results suggest that MAOA genotypes may participate dierentially in the regulation of dopamine and serotonin turnover rates under presumed steady state in the central nervous system. The results should be interpreted with caution until replicated because of the limited sample size. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Monoamine oxidase A gene; Monoamine metabolites (HVA; 5-HIAA; MHPG); Cerebrospinal ¯uid
1. Introduction Concentrations of the major monoamine metabolites (MM) 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in lumbar cerebrospinal ¯uid (CSF) have been used extensively as indirect measures of monoamine metabolism and turnover in the brain of humans. Studies of human twins indicate that CSF 5Part of this investigation has been presented at the 7th World Congress on Psychiatric Genetics, Monterrey, California, October 14±18, 1999. * Corresponding author. Tel.: +46-8-51772626; fax: +46-8346563. E-mail address: [email protected] (E.G. JoÈnsson). p
HIAA and HVA levels are under familial in¯uence of both genetic and environmental origin, while MHPG is under major genetic in¯uence (Oxenstierna et al., 1986). In rhesus monkeys signi®cant portions of CSF 5-HIAA, the major serotonin metabolite, HVA, the major dopamine metabolite, and MHPG, the major norepinephrine metabolite in the central nervous system, have been shown to be determined by genetic mechanisms (Higley et al., 1993). Monoamine oxidase (E.C. 1.4.3.4) is a mitochondrial enzyme involved in the degradation of biogenic amines including dopamine, serotonin, and norepinephrine. In humans two isozymes exist: monoamine oxidase A (MAOA), which preferentially deaminates serotonin and norepinephrine, and monoamine oxidase B (MAOB) catalyzing degradation of phenylethylamines and benzylamine
0022-3956/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 3 9 5 6 ( 0 0 ) 0 0 0 1 3 - 3
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(Bach et al., 1988; Cawthon et al., 1981; Donnelly and Murphy, 1977). With regard to dopamine, at least in man, both isoenzymes participate with some preponderance of MAOB (StenstroÈm et al., 1987). This makes both the MAO genes, located on chromosome Xp11.23±Xp11.4 (Kochersperger et al., 1986; Lan et al., 1989; Levy et al., 1989; Ozelius et al., 1988; Pintar et al., 1981), of interest in studies of mechanisms for regulation of monoamine turnover and metabolism. Recently a MAOA promoter variable number tandem repeat (VNTR) polymorphism has been reported (Deckert et al., 1999; Sabol et al., 1998). This VNTR consists of exact repeats of a 30-bp sequence. Two to ®ve exact repeats have been reported. Also, the four complete repeats are followed by a half repeat consisting of the ®rst 15 bp of the repeated motif, giving the following repeat nr: 2, 3, 3.5, 4, and 5. In transfection experiments both Deckert et al.(1999) and Sabol et al.(1998) have shown that alleles with 3.5 and 4 copies are transcribed more eciently than those with 3 copies. In the present study we have examined this MAOA VNTR polymorphism for possible relationships to concentrations of 5-HIAA, HVA, and MHPG in lumbar CSF from healthy Swedish volunteers. 2. Methods 2.1. Healthy human volunteers The investigation was carried out in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of the Karolinska Hospital, Stockholm. Informed consent of the subjects was obtained after the nature of the procedures had been fully explained. The characteristics and assessment of the majority of subjects (n = 66) participating in the present study have previously been described (JoÈnsson et al., 1996). In this study another 22 Caucasian individuals were added. Of these subjects 16 were initially drawn from a population register from Stockholm county council to take part in a study regarding CSF circulation including lumbar puncture (Oxenstierna et al., 1996). Otherwise these subjects were assessed in a similar way as the original 66 individuals, who were recruited predominantly among students or hospital sta. Lumbar puncture (LP) was performed in all subjects. Height was also recorded. Back length, de®ned as the distance between the external occipital protuberance and the insertion point of the lumbar needle with the subject in the lying position, was measured in 63 subjects. Eight to 19 years later a structured interview was performed by a psychiatrist (EJ) to assess psychiatric morbidity (DSM-III-R; American Psychiatric Association, 1987), somatic illness and presence of mental and nervous
system disorders among relatives. Subjects completed a questionnaire regarding smoking habits. Hospital records were obtained and examined for diagnosis. Genealogical data for antecedents up to the third degree were obtained from parish registers to assess the origin of the individuals. Subjects who reported any life time psychiatric disorder were excluded. Of the subjects 51 were men and 37 women. The age range at the time of the present study was 29±56 years, with a mean of 41 years. The mean age at LP was 27 years, age range 19±43. Thirty six were university graduates. Twenty one subjects had a family history of major mental illness de®ned as at least one ®rst or second degree relative with schizophrenia, schizoaective disorder, bipolar disorder, recurrent unipolar disorder, other non-organic psychosis, or who had committed suicide. Of the subjects 53 were or had been regular tobacco users, 18 were non smokers or had only used tobacco once or a few times in their life, while data were missing for 11 individuals. Of the women, 15 used oral contraceptives at LP, 20 did not, while data were missing for two individuals. Except for oral contraceptives all participants were drug free at LP. Genealogical data implicated that 89 and 5% of the genes originated in ancestors born in Sweden and Finland, respectively, and the remaining 6% were distributed over eight European countries. 2.2. CSF MM concentrations All subjects had at least 8 h of bed rest in the hospital, abstaining from food and smoking. CSF samples were obtained by LP between 8 and 9 a.m.with the subjects in the sitting (n = 41) or recumbent (n = 47) position. Samples of 12.5 ml CSF were drawn according to a standardized sampling procedure (Sedvall et al., 1980). Samples were stored at below ÿ208C and analyzed within 2 months. HVA, 5-HIAA, and MHPG concentrations were measured by mass fragmentography with deuterium labelled internal standards (Swahn et al., 1976). 2.3. Genotype analyses Venous blood was taken from all individuals into EDTA-containing tubes. DNA was isolated as previously described (Geijer et al., 1994). The 186±276 bp fragments containing the MAOA VNTR were ampli®ed using the following primers: forward ¯uorescently labelled 5'-GCC CAG GCT GCT CCA GAA A-3 ' and reverse 5 '-GAA CGG ACG CTC CAT TCG GA-3'. The PCR conditions were as follows: 200 mM dNTPs, 1 Taq Gold PCR buer II (Perkin Elmer), 2.5 mM Mg, 1 unit Taq Gold (Perkin Elmer), and 48 ng DNA, with an initial denaturating
E.G. JoÈnsson et al. / Journal of Psychiatric Research 34 (2000) 239±244
period at 958C for 10 min, followed by 35 cycles of 95 (45 sec), 62 (45 sec), and 628C (10 min). The PCR products were electrophoresed on 10% denaturing polyacrylamide gels on an ABI 373 DNA sequencer, and analysed using the ABI GENESCAN and GENOTYPER software. 2.4. Data analysis Subjects were grouped into those with or without 3.5/4 alleles, respectively, because the MAOA alleles containing 3.5 and 4 alleles have been shown to induce a more eective transcription than other alleles (Sabol et al., 1998). In a previous study, of the majority of the present sample, back length was found to be a signi®cant confounding variable for MM concentrations in CSF (JoÈnsson et al., 1996). To make it possible to correct MM for back length, back length values, based on the relationship between back length and height (r = 0.73, p < 0.0001), were constructed for those where only height had been measured (n = 26). Analysis of covariance (ANCOVA) was used to adjust for dierences in CSF levels due to back length. Because correcting MM concentrations for back length is still controversial (JoÈnsson et al., 1997), analyses were also performed with MM data uncorrected for back length. Analysis of variance (ANOVA) was used. Signi®cance level was de®ned as a p-value less than 0.05. Because of the exploratory nature of the investigation, no corrections were made for multiple comparisons. However, the reader is reminded of the increased potential for type I errors due to the large number of analyses performed (Grove and Andreasen, 1982). 3. Results The results shown in Table 1 demonstrate the stat-
241
istical data analysis performed on MM residuals corrected and uncorrected for back length. MM means and standard deviations (SD) are presented as uncorrected values. Four dierent alleles were detected in the present sample. In one women an allele with only two repeats was found. The allele frequencies were 0.01 (2 repeats), 0.31 (3 repeats), 0.02 (3.5 repeats), and 0.66 (4 repeats), respectively. Among men the allele frequencies were 0.22 (3 repeats), 0.02 (3.5 repeats), and 0.77 (4 repeats), respectively. In women the allele frequencies were 0.01 (2 repeats), 0.38 (3 repeats), 0.03 (3.5 repeats), and 0.58 (4 repeats), respectively, distributed on the following genotypes: 2/3 (3%), 3/3 (11%), 3/3.5 (3%), 3/4 (49%), 3.5/4 (3%), and 4/4 (32%). As the MAOA gene is located on the X chromosome, each gender was analysed separately. In women a relationship was indicated between the MAOA polymorphism and HVA-levels (F = 5.22, d.f.=1, p = 0.029 and F = 6.65, p = 0.015 uncorrected and corrected for back length, respectively). Women with genotypes including the 3.5 or 4 repeat alleles had higher HVA concentrations than women without these alleles. A similar relationship was obtained when the female subjects were analyzed for MAOA genotypes with regard to 5-HIAA levels. Women with genotypes including 3.5 or 4 repeat alleles displayed higher 5HIAA levels than those without these alleles (F = 7.47, d.f.=1, p = 0.010 and F = 7.31, p = 0.011 uncorrected and corrected for back length, respectively). However, no relationship was found in women between the MAOA polymorphism and MHPG levels. Women were also grouped into subjects only carrying alleles suggested to give rise to high transcription (genotypes 3.5/4 and 4/4), low transcription (2/3, 3/3), or subjects carrying both these types of alleles (3/3.5, 3/ 4). When compared vs CSF MMs the following results
Table 1 Comparisons (ANOVA) between monoamine oxidase A (MAOA) genotypes and homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) concentrations in lumbar cerebrospinal ¯uid of healthy volunteers HVA
5-HIAA
MHPG
Sex
Allele
n
Mean2SD (nmol/L)
Fa pa
Fb pb
Mean2SD (nmol/L)
Fa pa
Fb pb
Mean2SD (nmol/L)
Fa pa
Fb pb
Men
3.5/4 Other Genotype 3.5/4c Other 3.5/4+4/4 3/3.5+3/4 2/3+3/3
40 11
154261 208290
F = 3.43 p = 0.070
F = 5.33 p = 0.025
89235 108240
F = 1.04 p = 0.313
F = 2.47 p = 0.122
4227 4629
F = 0.85 p = 0.361
F = 1.54 p = 0.221
32 5 13 19 5
209272 133245 192253 221282 133245
F = 6.65 p = 0.015 F = 3.75 p = 0.034
F = 5.22 p = 0.029 F = 3.32 p = 0.048
113235 68215 106233 117237 68215
F = 7.31 p = 0.011 F = 4.14 p = 0.025
F = 7.47 p = 0.010 F = 4.21 p = 0.023
4226 3722 4126 4226 3722
F = 2.57 p = 0.118 F = 1.42 p = 0.256
F = 2.58 p = 0.117 F = 1.46 p = 0.246
Women Women
a
Statistical comparisons corrected for back length. Statistical comparisons uncorrected for back length. c Genotypes containing alleles 3.5 and/or 4.
b
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emerged: CSF HVA (F = 3.32, d.f.=2, p = 0.048 and F = 3.75, p = 0.034 uncorrected and corrected for backlength, respectively), CSF 5-HIAA (F = 4.21, d.f.=2, p = 0.023 and F = 4.14, p = 0.025 uncorrected and corrected for backlength, respectively), and MHPG (F = 1.46, d.f.=2, p = 0.246 and F = 1.42, p=0.256 uncorrected and corrected for backlength, respectively). Without correction for back length there was a relationship in men (F = 5.33, d.f.=1, p = 0.025) with lower CSF HVA-levels in subjects with the 3.5 or 4 repeat alleles as compared to those lacking these alleles. However, after correction for back length this relationship was reduced to a trend (F = 3.43, p = 0.070). No relationships was found in men between the MAOA polymorphism and CSF 5-HIAA and MHPG concentrations, respectively. 4. Discussion To our knowledge the present publication is the ®rst comparing MAOA genotypes and lumbar CSF MM in healthy human subjects. In women, relationships were found between MAOA genotypes and CSF concentrations of HVA and 5-HIAA. Women with genotypes containing alleles with 3.5 and/or 4 repeats, reported to induce a more eective monoamine oxidase transcription (Deckert et al., 1999; Sabol et al., 1998), were found to have higher levels of HVA and 5-HIAA. A similar tendency was also found with regard to MHPG in women, although this dierence was not signi®cant. It would seem reasonable that a higher amount of a converting enzyme may contribute to higher levels of a resulting degradation product and, furthermore, treatment with an MAOA inhibitor results in a decrease in CSF levels of monoamine transmitter metabolites (Major et al., 1979). The experiments by Sabol et al. (1998) showed that the MAOA activities diered considerably when the dierent MAOA gene promoter VNTR alleles were transfected to cell lines. Post mortem studies on human brain tissues, on the other hand, indicate that variation in MAOA activity is remarkably low (Fowler et al., 1980). Thus, it remains to be shown that the present MAOA gene polymorphism is associated with variation in enzyme activity in vivo. In men no signi®cant relationships emerged, although a trend in the opposite direction as compared to women was found between MAOA alleles and HVA levels. This may indicate that there are marked dierences between men and women with regard to the strength of these relationships. There have been reports of gender dierences with regard to CSF monoamine metabolites (Blennow et al., 1993; Constantino and Murphy, 1996; Nordin et al., 1995). If there is a real dierence between men and
women with regard to the relationships between MAOA genotypes and monoamine metabolite levels this may indicate certain confounding factors which dier between the sexes. In¯uence of steroids has been discussed as one such potential confounder (Best et al., 1992; JoÈnsson et al., 1997). Interstingly, in the present sample female users of contraceptives had signi®cantly lower CSF-levels of 5-HIAA (t = 2.40, d.f.=33, p = 0.023) and MHPG (t = 2.25, d.f.=33, p = 0.031) than non-users. These dierences were not signi®cantly in¯uenced by back length (data not shown). Similar dierences with regard to CSF MHPG, but not 5HIAA levels have previously been reported (Eklundh et al., 1994; TraÈskman-Bendz, 1980). However, we did not ®nd any MAOA genotype dierence between users and non-users of contraceptives (Fisher's exact test p = 0.141) indicating that the relationships between MAOA genotypes and monoamine metabolite levels are not signi®cantly in¯uenced by the use of contraceptives. Tobacco use has been suggested to signi®cantly in¯uence CSF HVA (Geracioti Jr et al., 1999) and also to considerably inhibit the activity of monoamine oxidase (Fowler et al., 1996a,b). In the present sample, however, no signi®cant dierences were found when use or non-use of tobacco were compared vs MAOA allele/genotype constalletions or levels of HVA, 5HIAA, or MHPG in each sex separately or with the two sexes combined, respectively (data not shown). This result, together with the previously reported small variation in enzyme activity in post mortem brain tissue (Fowler et al., 1980), may indicate that the associations found are not the result of a direct cause relationship. An alternative explanation would be that the in¯uence of the dierent MAOA alleles on the expression of enzyme activity during prenatal development will induce life-long eects on brain monoamine systems, similar to those induced in rats by prenatal MAOA inhibition (Whitaker-Azmitia et al., 1994). The signi®cant CSF HVA and 5-HIAA dierences with regard to MAOA genotypes in the present study may be chance ®ndings. The sample size is limited. Also considering the many variables tested, type I errors can be expected to emerge. There were six primarily independent investigations performed (genotypes of one polymorphism vs concentrations of three monoamine metabolites in two sexes) in the total sample. This gives a p-value of less than 0.008 to be considered signi®cant using Bonferroni's correction. Thus, applying this correction in the total sample no relationship would remain signi®cant. This means that until the present results are replicated in independent investigations, they must be regarded as preliminary and should be interpreted with caution. Dopamine, serotonin and norepinephrine transmission have been implicated in aective disorder and
E.G. JoÈnsson et al. / Journal of Psychiatric Research 34 (2000) 239±244
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