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MAOA haplotypes associated with thrombocyte-MAO activity

MAOA haplotypes associated with thrombocyte-MAO activity Background: The aim was to ascertain whether thrombocyte MAO (trbc-MAO) activity and depressed state are genetically associated with the MAO locus on chromosome X (Xp11.3 – 11.4). We performed novel sequencing of the MAO locus and validated genetic variants found in public databases prior to constructing haplotypes of the MAO locus in a Swedish sample (N = 573 individuals). Results: Our results reveal a profound SNP desert in the MAOB gene. Both the MAOA and MAOB genes segregate as two distinct LD blocks. We found a significant association between two MAOA gene haplotypes and reduced trbc-MAO activity, but no association with depressed state. Conclusion: The MAO locus seems to have an effect on trbc-MAO activity in the study population. The findings suggest incomplete X-chromosome inactivation at this locus. It is plausible that a gene-dosage effect can provide some insight into the greater prevalence of depressed state in females than males. locus were found among individuals of northern Euro- Background Monoamine oxidase A (MAOA) and B (MAOB) are pean ancestry [5]. enzymes that deaminate monoamines such as serotonin, dopamine and noradrenaline. The genes encoding MAOA Both enzymes are localized in the outer mitochondrial and B are located on the X chromosome in a tail-to-tail membrane [7]. They are also present in glial cells [8], orientation and separated by approximately 20 kilobases although MAOA is less expressed than MAOB [9]. The (kb) [1,2]. Although MAOA and MAOB span 65 kb and enzymes differ in their expression patterns not only 116 kb, respectively, both genes display a high degree of peripherally in the body but also in the central nervous homology and most certainly have a common ancestry system (CNS) [10]. MAOB is the only form that is [3]. The frequencies of confirmed polymorphisms in the expressed in human blood cells. MAOA is primarily two genes vary widely among different ethnic groups [4- expressed in catecholaminergic neurons in the human 6]. Only two common haplotype variants of the MAOA brain [10,11], whereas MAOB is expressed in serotonergic Page 1 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 [10] and histaminergic neurons [8]. The two MAO- two additional variants further down stream with a minor enzymes also differ on substrate preferences; MAOA pref- allele frequency greater than 1%. Both the recorded and erentially metabolizes serotonin and norepinephrine most distal variants showed complete LD with each other, while MAOB has a much higher affinity for phenylethyl- therefore only the recorded variant was chosen for further amine [12,13] and benzylamine [14]. analysis. Thrombocyte-MAO activity (Trbc-MAO) has been associ- The genotyping error rate was calculated at 0,4% through ated with cerebrospinal fluid (CSF) levels of serotonin males scoring as heterozygotes and from MZ twins where metabolites in humans [15] and is higher in women than both twins in a pair were genotyped. These errors could men [16-18]. This difference has been speculated to be an not be scored differently from the sequence and therefore effect of sex steroids altering the enzyme's activity or a most likely reside in the handling of samples, e.g. contam- matter of escaped X-inactivation [19]. The proportion of ination or labelling error. variance in trbc-MAO activity explained by genetic factors (its heritability) in a Swedish population is 77% [20]. In addition to resequencing the upstream regions, we gen- Trbc-MAO activity is weakly associated with a C/T poly- otyped reported SNPs in the remainder of the gene clus- morphism in intron 13 of the MAOB gene in a Swedish ters by Pyrosequencing. Six of the previously reported population [21] and is also influenced by smoking and SNPs could not be confirmed as polymorphic specific medications; smokers can have a 30–40% lower (rs1014876, rs3027464, rs6324, rs1040398 and two SNPs trbc-MAO activity than non-smokers [22]. Trbc-MAO reported by Balciuniene et al.) The remaining nine poly- activity is also associated with several psychiatric syn- morphic variants; one in the Norrie gene (rs766117), four dromes [23], personality traits and mood disorders e.g. SNPs in MAOB (rs1181252, rs2283729, rs3027452 and [24-28]. rs1799836) and four SNPs in MAOA (rs1801291, rs979605, rs6323, rs388863) were sequenced in the total In the present study we address issues concerning genetic sample. variation in MAOA and MAOB genes, activity levels of trbc-MAO, and associations with depressed state. Genetic The LD map (Figure 2) displays a clear structure of the variation was analyzed by sequencing the regulatory MAO locus with strong LD across the MAOA gene in a dis- region of both MAOA and MAOB, and validating SNPs tinct block spanning approximately 65 kb. The MAOB reported in public databases. We used multiple SNPs cov- gene also displays a similar block-like structure, although ering the MAO gene locus to generate haplotypes on a the pattern of LD is not as robust as for MAOA. This is per- population level. Finally, we investigated associations haps due to the inconsistencies in allele frequencies across between depressed state and trbc-MAO activity and MAOB. Interestingly, weak LD is observed at the tail ends genetic variants in the MAO locus in a large elderly Swed- between the two MAO loci. ish population. Furthermore, because there was no LD between the Norrie gene variant, located >66 kb upstream of MAOB, and any Results Trbc-MAO activity and depressed state other variant in the MAO region, we decided not use this We found a clearly significant difference between males variant further in the haplotype assessment. Modest devi- (mean; 10.7) and females (mean; 12.1) (t = 4.69; p ≤ ations from Hardy-Weinberg equilibrium were noted in 0,0001), as well as between smokers and non-smokers in rs766117 in the Norrie gene (p = 0,022) and rs979605 in mean trbc-MAO activity (t = 5,86; p =< 0,0001). Smokers intron 10 of the MAOA gene (p = 0,028). This could reflect showed a 23% lower trbc-MAO activity compared to non- the underlying LD structure [29], as demographic influ- smokers. Females with a depressed state showed a signifi- ences would act over larger regions [30]. However to clar- cantly higher mean trbc-MAO activity than unaffected ify this, a denser set of SNPs would need to be genotyped. females (t = 2,02; p = 0,04). In the male population we could identify five distinct hap- Genetic variants and haplotype construction lotypes in the MAOB gene and four in the MAOA gene Approximately 4.5 kb of both the MAOA and MAOB gene with frequencies ≥1% (Figure 1.). When analyzing the promotor, including the first exons, totaling 9 kb, were MAO locus as one large block using eight SNPs, we found sequenced from a total of 148 X chromosomes. Power to ten distinct locus haplotypes with a frequencies ≥1% (data discover SNPs with frequencies greater than 1% and 3% not shown). In the female population, "PHASE" assem- for this sample size was 77% and 100%, respectively. No bled identical higher frequency haplotypes as were identi- variants were found in the MAOB gene. In contrast, one fied in the male sample, with minor discrepancies in previously reported variation was confirmed (rs3788863) lower frequency haplotypes due to unknown phase (Fig- for the MAOA gene, lying within the first intron, as well as ure 1). Page 2 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Genetic structure of the MAO locus Figure 1 Genetic structure of the MAO locus. Haplotype and common allele frequencies in the total sample. dbSNP rs numbers for all genotyped SNPs are presented with major allele frequencies. Haplotypes frequencies illustrated for MAOA and B separately as well as the genes combined (See Text). NDP was not used in the haplotype frequency estimations. Associations with SNPs ses of the entire MAO locus and trbc-MAO activity did not In the total sample, no single variant of any of the individ- reveal any significant findings (data not shown). We ual SNPs was associated with trbc-MAO activity. However, could not find any significant associations between in females the C/C and C/T genotypes of rs979605 in the depressed state and any specific haplotype in men or MAOA gene were associated with a significant decrease in women (Table 2). trbc-MAO activity, (-2,9; CI 95%: -5,2 – -0,6) and (-2,4; CI 95%: -4,7 – -0,1) respectively. When the model was analyzed without genetic informa- tion, males have a significantly lower risk for being Analyzed by gender, depressed state was associated with affected with depressed state compared to women (OR = the A-allele of MAOB SNP rs1181252 in males (OR = 4,5; 0,5). This gender effect may be explained by the genetic CI 95%: 1,0 – 21,7) and both GG and GA of rs766117 information (even though no associations were found (OR = 2,2; CI 95%: 1,1 – 4,3) in females. It should be with any of the haplotypes), because the risk for depressed noted that the "A" allele of rs1181252 only had a popula- state due to the male gender is differs in the analyses of the tion frequency of 6%. MAOA locus (OR = 1,4; non-significant) and the MAOB locus, where the estimate is similar to the model without Associations with haplotypes genetic information. There was no association between any of the MAOB hap- lotypes and trbc-MAO activity. Two MAOA haplotypes, A1 Interestingly, in females all MAOB homozygote haplo- and A3, both sharing identical alleles at the three first hap- types displayed greater odds ratios with depressed state lotype positions (CCA-) (Figure 1), were associated with a than that for heterozygotes (Table 2), indicating an addi- significant decrease in trbc-MAO activity (Table 1). Analy- tive effect. The same was true for MAOA (Table 2). Page 3 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 LD map Figure 2 LD map. Pair-wise LD map with one individual from each female pair (N = 356). D' is shown below the diagonal and ∆ above the diagonal. Color code D': Red: ≥0,8 Orange: 0,5–0,8 Yellow: 0,3–0,5 White: <0,3. Color code ∆ : Red: ≥0,30 Orange: 0,1– 0,30 Yellow: 0,05–0,1 White: <0,05. state. On the other hand, there was an interesting, Discussion Monoamine oxidase A and B constitute two important although not significant dose-response effect of haplo- molecules in the human body in general and in the types displayed in women, with greater odds ratios in central nervous system (CNS) in particular. Numerous homozygotes than heterozygotes. studies suggest a contribution of these two mitochondrial enzymes to complex human behaviors [26-28,31-33]. In Considering the size and importance of the MAO locus, the present study we searched the MAO locus for novel relatively few polymorphic sites have been verified. We genetic variants and evaluated the genetic and haplotype observed two new variants through sequence screening a structure in a Swedish population. We also assessed asso- partial region of MAOA intron 1, but in MAOB neither the ciations between trbc-MAO activity and depressed state, previously reported nor any novel variants were found in and their respective associations with the genetic structure the areas sequenced. It is surprising that so few SNPs were of the MAO locus. The key findings of this study are first: discovered given our power to detect variants with very the profound lack of variation at functional regions of the low frequencies. SNP deserts have been previously noted two MAO genes and a pattern of two distinct genetic LD on the q arm of the X chromosome [34]. Gilad and col- blocks, one for each gene. Second: we replicated the gen- leagues [4] have described similar features across MAOA, der differences in trbc-MAO activity and demonstrated an where extensive LD and low nucleotide diversity suggest association between trbc-MAO activity and depressed recent action by population structure forces and perhaps a state in women. Third: two MAOA haplotype variants recent positive selection sweep [35]. Although we could were associated with decreased trbc-MAO activity not evaluate the influence of such forces, evidence of although we could not replicate a previously reported strong LD and the lack of decay across MAOA in our Swed- genetic association between the MAOB gene and trbc- ish sample complement these previous findings. Linkage MAO activity. Fourth: we could not find any significant disequilibrium decays rapidly between the two MAO associations between the genetic variants and depressed genes (separated by approximately 20 kb). Perhaps selec- Page 4 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Table 1: Associations between MAO haplotypes and trbc-MAO activity, reported as unit change in mean trbc-MAO activity per allele and controlling for gender and smoking status. Total sample N = 340 Males N = 156 Females N = 184 per allele Hemizygous per allele Haplotypes Estimates (Unit change in mean trbc-MAO activity per allele) B1 -0,38 (-1,3 – 0,5) 0 (ref) -0,3 (-1,1 – 0,5) B2 -0,63 (-1,8 – 0,5) -0,08 (-1,6 – 1,5) -0,4 (-1,5 – 0,7) B3 -0,18 (-1,6 – 1,3) -0,8 (-2,9 – 1,2) -0,2 (-1,5 – 1,0) B4 -1,3 (-3,5 – 0,8) 0,7 (-2,8 – 4,3) -1,0 (-2,8 – 0,8) B5 -1,7 (-5,1 – 1,6) NA -0,7 (-3,8 – 2,4) Male gender -2,1 (-3,3 – -0,9)* Non-smokers 2,3 (1,1 – 3,5)* 1,5 (0,2 – 2,8) 3,5 (2,0 – 5,0) A1 -1,1 (-1,9 – -0,3)* -1,8 (-3,2 – -0,5)* -1,0 (-1,7 – -0,3)* A2 0,1 (-0,9 – 1,2) 0 (ref) 0,6 (-0,4 – 1,5) A3 -3,1 (-6,1 – -0,14)* -2,3 (-5,8 – 1,2) -4,1 (-7,4 – -0,7)* A4 -0,02 (-3,8 – 3,7) NA -0,5 (-3,7 – 2,7) Male gender -2,3 (-3,5 – -1,1)* Non-smokers 2,4 (1,1 – 3,6)* 1,4 (0,03 – 2,8) 3,4 (1,9 – 4,8) tion is in action much more locally than would be other hand, it is possible that the MAOA locus holds cis- expected in each MAO gene, both separated by regions of acting regulatory elements affecting MAOB expression. higher recombination than that within each gene. Another possible explanation could be that one or several single-base variants affected by methylation cause Previous studies have indicated that the MAOA gene may changes in the expression pattern [40]. harbour relatively few haplotypes within a block structure [5]. We observed similar results here with two haplotypes Our study is based on a relatively large population-based encompassing 95% of the haplotypic variation. We found sample of normally aging adults, although it is not with- similar results for the MAOB gene, with a distinct block out its limitations. We have controlled for smoking, but structure in which three haplotypes explain 93% of the were unable to do so for intake of certain medications. variation. So few haplotypes over such long distances The study sample was included in a larger study where have been observed previously (McCarthy et al, manu- associations between depressed state and the serotonin script) and are proposed signatures of selection and pop- receptor 2A and the serotonin transporter were evaluated ulation substructure on the X chromosome [36,37]. [41]. The influence of these genes has not been corrected for in the analysis. A previous Swedish association between the MAOB gene and trbc-MAO activity [21] could not be replicated nor Conclusion distinctly refuted, as we found a small non-significant Good et al [19] demonstrated that trbc-MAO activity is effect of the same allele in males. However, none of the related to the number of X chromosomes. We replicated a haplotype blocks carrying this allele could strengthen or significant difference in trbc-MAO activity between males support this effect, suggesting that this allele is not in high and females reported by others e.g. [17]. The findings sug- LD with a larger region of the MAOB gene. gest incomplete X chromosome inactivation at this locus and are consistent with other findings of genes escaping Two MAOA haplotypes (A1 and A3) showed a significant inactivation on the X chromosome [42,43]. It has been association with reduced trbc-MAO activity. Both haplo- hypothesized that this dosage imbalance between males types shared the initial sequence variants [CCA], but var- and females might be crucial for gender characteristics ied at the fourth allele [T/C]. Given that only MAOB is [19,44]. Recently it was demonstrated that a number of expressed in platelets there is no clear explanation for this genes, including MAOA, escape X-inactivation [45]. Fur- finding. Given the minor kinetic differences between thermore, the X-inactivation pattern, which shows a sub- platelet and brain MAO-B [38] and the correlation of stantial heritability [46], increases in the elderly. Although MAOB and MAOA levels in regions of the brain [39], this we could not find a significant association between vari- association may reflect MAOA activity in the brain. On the ants of MAOB or MAOA and depressed state in this popu- Page 5 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Table 2: MAO haplotypes and depressive state, reported as odds ratios per allele. Without genetic information in the model male gender was significant [OR: 0,5 (0,3 – 0,8)] for depressive state. *Homozygote compared to heterozygote. MAO haplotypes and depressive state Total sample N = 573 Males N = 239 Females N = 334 per allele Hemizygous per allele Homozygotes* Odds Ratio with 95% CI B1 1,2 (0,6 – 2,5) 1,0 (ref) 1,4 (0,6 – 2,9) 1,3 p = 0,57 B2 1,5 (0,7 – 3,3) 1,7 (0,8 – 3,6) 1,5 (0,6 – 3,3) 1,2 p = 0,80 B3 1,3 (0,5 – 3,0) 0,7 (0,3 – 1,7) 1,7 (0,7 – 4,2) 1,9 p = 0,51 B4 2,0 (0,8 – 5,2) 3,7 (0,7 – 18,7) 2,0 (0,7 – 5,3) 1,7 p = 0,59 B5 0,5 (0,1 – 2,8) 2,4 (0,4 – 14,5) 0,3 (0,04 – 2,5) NA Male gender 0,7 (0,3 – 1,5) A1 3,0 (0,8 – 12,2) 1,0 (ref) 2,2 (0,6 – 8,4) 5,5 p = 0,08 A2 2,5 (0,6 – 10,6) 0,9 (0,4 – 1,8) 1,7 (0,4 – 7,1) 1,3 p = 0,80 A3 2,8 (0,7 – 11,4) 1,2 (0,3 – 4,4) 1,7 (0,4 – 6,9) NA A4 3,2 (0,6 – 18,6) NA 2,8 (0,4 – 17,3) NA Male gender 1,4 (0,3 – 6,0) lation, we found an interesting dose-response effect in blood donors were randomly selected from a larger sam- women, with a higher risk for depressed state with ple set collected to study MAOB regulation. All were homozygosity. Whether levels of trbc-MAO activity are between the ages of 20 to 40 years and non-smokers. correlated with the number of X chromosomes and whether this might be linked to the higher prevalence of This study was reviewed and approved by the Ethics Com- depressive symptoms in females deserves further investi- mittee of the Karolinska Institute, the Swedish Data gation. Nevertheless it is plausible that a partially doubled Inspection Board, and the IRBs at the University of South- gene activity on the X chromosome can explain difference ern California and the Pennsylvania State University. All in prevalence of depressive state in men and women. subjects provided informed consent. DNA and trbc-MAO activity Methods Participants DNA samples were available from 573 twins. Trbc-MAO The participants were taken from a longitudinal twin activity measures were available from 565 twins. The trbc- study of aging, the Swedish Adoption/Twin Study of MAO activity is expressed as nmoles of 2-phenylethyl- platelets. Trbc- Aging (SATSA) with up to five occasions of measurement amine oxidized per minute and per 10 [47]. SATSA is a sub-sample of the population based MAO activity measures have previously been described in Swedish Twin Registry [48]. All participants are Caucasian detail [20]. and born in Sweden. For the present analyses we selected Depressed state all individuals who participated in an in-person testing session during which questionnaires were administered Depressive symptoms were measured with the Center for and a blood sample was drawn. The mean age of the sam- Epidemiologic Studies Depression Scale (CES-D), a 20- ple was 61,3 years at the time of testing. Twenty two per- item self-report instrument developed for use in the com- cent of the participants were current smokers; 35% of the munity and well established for use with older adults males and 15% of the females. [49,50]. The scale has been shown to have minimal over- lap with physical illness [51] and assesses current symp- Zygosity was initially based on self-reports of similarity toms during the past week. Respondents scoring 16 or and confirmed by serological analyses and comparisons higher on the CES-D scale are considered to have a clini- of up to 10 DNA markers. cally relevant depressed state. In this study population of 574 participants, 144 were classified as having a depressed For preliminary screening of the promoter, the first exon state, 17.9% of the males and 30.2% of females. and intron regions for novel variants, 94 Swedish male Page 6 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Genotyping & sequencing matics.org/macroshack/programs/PHARE) to create input Approximately 4.5 kb of each gene was initially sequenced files for "PHASE" [59,60] to construct female haplotypes. in search of novel SNPs in both MAOA and MAOB, first in 94 Swedish males and later 45 twins with CES-D scores We used linear regression to estimate the association (36 males and 9 females). Power to detect minor allele fre- between trbc-MAO activity and genotypic information quencies (q) between 1 and 5% was determined as by using a generalized estimating equation (GEE) approach Glatt et al. [52], 1-(1-q) where N is the number of chro- and alternating logistic regression (ALR) [61] to estimate mosomes. Amplification and nested sequencing primers the association between depressed state and genotypic were designed with the CPrimers programme from Gen- information. We first modeled the association between bank entry GI:8671203 containing the promoter, coding single SNPs and each of the two outcomes and then mod- exon 1 and flanking intronic sequence of MAOA (~5.0 kb, eled the association between haplotype constructs and the nucleotides 46490–51454) and Genbank entry two outcomes. All estimates were adjusted for current GI:2440066 spanning the same characterized sequences smoking status. We estimated both dominance and co- of MAOB (~4 kb nucleotides 35033–39021). dominance models. Explanatory variables in the domi- nance models were binary whereas in the co-dominance Direct sequencing reactions were performed using models they were coded as the number of reference alleles DYEnamic ET Dye Terminator Cycle Sequencing Kit (i.e., 0, 1, or 2 for females and 0 or 1 for males). The (Amersham Biosciences) and separated using a Megabace parameter estimates for the co-dominance models repre- 1000. Reads were base called with Phred [53], assembled sent the change in the outcome (trbc-MAO activity or using Phrap and viewed using Consed Version 13 [54]. All odds of being in a depressed state) per affected allele. Due SNPs were documented and cross validated with dbSNP at to the continuous nature of the trbc-MAO measure, only NCBI. one individual from each complete twin pair and single participating individuals were analyzed (N = 340). Twelve SNPs identified from public databases (dbSNP at Among females we also estimated the effect of being NCBI) and two novel SNPs previously reported (introns 3 homozygote compared to heterozygote. If the co-domi- and 10 of MAOB) a Swedish sample [5] were sequenced nance model is a good fit to the data then these estimates in 95 participants (142 chromosomes) by Pyrosequencing should be similar to the "per allele" estimates from the co- to confirm their presence in this population. For Pyrose- dominance model. All statistical analyses were performed quencing, either the forward or the reverse primer in each in SAS 8.01 using GENMOD procedure (SAS Institute Inc. primer pair was biotinylated. Sequencing primers with a Cary, NC). length of 14 and 18 bases were placed within one base of the SNP. The PCR reaction was performed in a 50 µl reac- Authors' contributions tion volume, containing 5 ng of genomic DNA, 10 pmoles MJ: Design of the study, performed data analysis and of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl and interpretation of data. Carried out the molecular genetic 1.5 U of Taq. Thermal cycling was performed in a PTC-225 studies (genotyping) and drafted the manuscript. DNA machine (MJ Research Inc., Cambridge, MA, USA) at 95°C for 5 min followed by 50 cycles of 95°C for 30 s, 45 SM: Participated in the design of the study. Carried out the s of annealing at an optimized temperature, followed by molecular genetic studies (sequencing), sequence align- 72°C for 30 s and a final extension of 5 min at 72°C. The ment and critically revised the manuscript. biotinylated PCR product was immobilized onto strepta- vidin-coated sepharose beads and DNA strands were sep- PFS: Participated in the design of the study and critically arated by denaturation with 0.2 M NaOH. The revised the manuscript for important intellectual content. pyrosequencing reaction was performed on a PSQ96™ Instrument from Pyrosequencing AB (Uppsala, Sweden) PD: Planed and performed the statistical analysis. as described by [55,56]. Detailed primer and assay infor- mation are available upon request. BA: Participated in the design of the study and critically revised the manuscript. Statistical analysis Male haplotypes could be extrapolated directly since the LO: Substantially revised the manuscript for important MAO locus is located on the X chromosome and males are intellectual content. thereby hemizygous. Female bi-allelic haplotypes were estimated using an EM algorithm (Sham 1998) and the MS: Participated in the design of the study and critically pair-wise LD measures D' [57] and ∆ [58]. We used revised the manuscript. "PHARE" (by David G Cox, available at http://bioinfor Page 7 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 15. Oreland L, Wiberg A, Asberg M, Traskman L, Sjostrand L, Thoren P, NLP: Participated in the design of the study and substan- Bertilsson L, Tybring G: Platelet MAO activity and monoamine tially revised the manuscript for important intellectual metabolites in cerebrospinal fluid in depressed and suicidal content. patients and in healthy controls. Psychiatry Res 1981, 4:21-29. 16. Bridge TP, Soldo BJ, Phelps BH, Wise CD, Francak MJ, Wyatt RJ: Platelet monoamine oxidase activity: demographic charac- All authors read and approved the final manuscript. teristics contribute to enzyme activity variability. J Gerontol 1985, 40:23-28. 17. 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Stephens M, Smith NJ, Donnelly P: A new statistical method for cited in PubMed and archived on PubMed Central haplotype reconstruction from population data. Am J Hum Genet 2001, 68:978-989. yours — you keep the copyright 61. Carey VJ, Zeger SL: Modelling multivariate binary data with BioMedcentral alternating logistic regressions. Biometrica 1993:517-526. Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Genetics Springer Journals

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Springer Journals
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Copyright © 2005 by Jansson et al; licensee BioMed Central Ltd.
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Life Sciences; Life Sciences, general; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics & Genomics; Genetics and Population Dynamics
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Abstract

Background: The aim was to ascertain whether thrombocyte MAO (trbc-MAO) activity and depressed state are genetically associated with the MAO locus on chromosome X (Xp11.3 – 11.4). We performed novel sequencing of the MAO locus and validated genetic variants found in public databases prior to constructing haplotypes of the MAO locus in a Swedish sample (N = 573 individuals). Results: Our results reveal a profound SNP desert in the MAOB gene. Both the MAOA and MAOB genes segregate as two distinct LD blocks. We found a significant association between two MAOA gene haplotypes and reduced trbc-MAO activity, but no association with depressed state. Conclusion: The MAO locus seems to have an effect on trbc-MAO activity in the study population. The findings suggest incomplete X-chromosome inactivation at this locus. It is plausible that a gene-dosage effect can provide some insight into the greater prevalence of depressed state in females than males. locus were found among individuals of northern Euro- Background Monoamine oxidase A (MAOA) and B (MAOB) are pean ancestry [5]. enzymes that deaminate monoamines such as serotonin, dopamine and noradrenaline. The genes encoding MAOA Both enzymes are localized in the outer mitochondrial and B are located on the X chromosome in a tail-to-tail membrane [7]. They are also present in glial cells [8], orientation and separated by approximately 20 kilobases although MAOA is less expressed than MAOB [9]. The (kb) [1,2]. Although MAOA and MAOB span 65 kb and enzymes differ in their expression patterns not only 116 kb, respectively, both genes display a high degree of peripherally in the body but also in the central nervous homology and most certainly have a common ancestry system (CNS) [10]. MAOB is the only form that is [3]. The frequencies of confirmed polymorphisms in the expressed in human blood cells. MAOA is primarily two genes vary widely among different ethnic groups [4- expressed in catecholaminergic neurons in the human 6]. Only two common haplotype variants of the MAOA brain [10,11], whereas MAOB is expressed in serotonergic Page 1 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 [10] and histaminergic neurons [8]. The two MAO- two additional variants further down stream with a minor enzymes also differ on substrate preferences; MAOA pref- allele frequency greater than 1%. Both the recorded and erentially metabolizes serotonin and norepinephrine most distal variants showed complete LD with each other, while MAOB has a much higher affinity for phenylethyl- therefore only the recorded variant was chosen for further amine [12,13] and benzylamine [14]. analysis. Thrombocyte-MAO activity (Trbc-MAO) has been associ- The genotyping error rate was calculated at 0,4% through ated with cerebrospinal fluid (CSF) levels of serotonin males scoring as heterozygotes and from MZ twins where metabolites in humans [15] and is higher in women than both twins in a pair were genotyped. These errors could men [16-18]. This difference has been speculated to be an not be scored differently from the sequence and therefore effect of sex steroids altering the enzyme's activity or a most likely reside in the handling of samples, e.g. contam- matter of escaped X-inactivation [19]. The proportion of ination or labelling error. variance in trbc-MAO activity explained by genetic factors (its heritability) in a Swedish population is 77% [20]. In addition to resequencing the upstream regions, we gen- Trbc-MAO activity is weakly associated with a C/T poly- otyped reported SNPs in the remainder of the gene clus- morphism in intron 13 of the MAOB gene in a Swedish ters by Pyrosequencing. Six of the previously reported population [21] and is also influenced by smoking and SNPs could not be confirmed as polymorphic specific medications; smokers can have a 30–40% lower (rs1014876, rs3027464, rs6324, rs1040398 and two SNPs trbc-MAO activity than non-smokers [22]. Trbc-MAO reported by Balciuniene et al.) The remaining nine poly- activity is also associated with several psychiatric syn- morphic variants; one in the Norrie gene (rs766117), four dromes [23], personality traits and mood disorders e.g. SNPs in MAOB (rs1181252, rs2283729, rs3027452 and [24-28]. rs1799836) and four SNPs in MAOA (rs1801291, rs979605, rs6323, rs388863) were sequenced in the total In the present study we address issues concerning genetic sample. variation in MAOA and MAOB genes, activity levels of trbc-MAO, and associations with depressed state. Genetic The LD map (Figure 2) displays a clear structure of the variation was analyzed by sequencing the regulatory MAO locus with strong LD across the MAOA gene in a dis- region of both MAOA and MAOB, and validating SNPs tinct block spanning approximately 65 kb. The MAOB reported in public databases. We used multiple SNPs cov- gene also displays a similar block-like structure, although ering the MAO gene locus to generate haplotypes on a the pattern of LD is not as robust as for MAOA. This is per- population level. Finally, we investigated associations haps due to the inconsistencies in allele frequencies across between depressed state and trbc-MAO activity and MAOB. Interestingly, weak LD is observed at the tail ends genetic variants in the MAO locus in a large elderly Swed- between the two MAO loci. ish population. Furthermore, because there was no LD between the Norrie gene variant, located >66 kb upstream of MAOB, and any Results Trbc-MAO activity and depressed state other variant in the MAO region, we decided not use this We found a clearly significant difference between males variant further in the haplotype assessment. Modest devi- (mean; 10.7) and females (mean; 12.1) (t = 4.69; p ≤ ations from Hardy-Weinberg equilibrium were noted in 0,0001), as well as between smokers and non-smokers in rs766117 in the Norrie gene (p = 0,022) and rs979605 in mean trbc-MAO activity (t = 5,86; p =< 0,0001). Smokers intron 10 of the MAOA gene (p = 0,028). This could reflect showed a 23% lower trbc-MAO activity compared to non- the underlying LD structure [29], as demographic influ- smokers. Females with a depressed state showed a signifi- ences would act over larger regions [30]. However to clar- cantly higher mean trbc-MAO activity than unaffected ify this, a denser set of SNPs would need to be genotyped. females (t = 2,02; p = 0,04). In the male population we could identify five distinct hap- Genetic variants and haplotype construction lotypes in the MAOB gene and four in the MAOA gene Approximately 4.5 kb of both the MAOA and MAOB gene with frequencies ≥1% (Figure 1.). When analyzing the promotor, including the first exons, totaling 9 kb, were MAO locus as one large block using eight SNPs, we found sequenced from a total of 148 X chromosomes. Power to ten distinct locus haplotypes with a frequencies ≥1% (data discover SNPs with frequencies greater than 1% and 3% not shown). In the female population, "PHASE" assem- for this sample size was 77% and 100%, respectively. No bled identical higher frequency haplotypes as were identi- variants were found in the MAOB gene. In contrast, one fied in the male sample, with minor discrepancies in previously reported variation was confirmed (rs3788863) lower frequency haplotypes due to unknown phase (Fig- for the MAOA gene, lying within the first intron, as well as ure 1). Page 2 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Genetic structure of the MAO locus Figure 1 Genetic structure of the MAO locus. Haplotype and common allele frequencies in the total sample. dbSNP rs numbers for all genotyped SNPs are presented with major allele frequencies. Haplotypes frequencies illustrated for MAOA and B separately as well as the genes combined (See Text). NDP was not used in the haplotype frequency estimations. Associations with SNPs ses of the entire MAO locus and trbc-MAO activity did not In the total sample, no single variant of any of the individ- reveal any significant findings (data not shown). We ual SNPs was associated with trbc-MAO activity. However, could not find any significant associations between in females the C/C and C/T genotypes of rs979605 in the depressed state and any specific haplotype in men or MAOA gene were associated with a significant decrease in women (Table 2). trbc-MAO activity, (-2,9; CI 95%: -5,2 – -0,6) and (-2,4; CI 95%: -4,7 – -0,1) respectively. When the model was analyzed without genetic informa- tion, males have a significantly lower risk for being Analyzed by gender, depressed state was associated with affected with depressed state compared to women (OR = the A-allele of MAOB SNP rs1181252 in males (OR = 4,5; 0,5). This gender effect may be explained by the genetic CI 95%: 1,0 – 21,7) and both GG and GA of rs766117 information (even though no associations were found (OR = 2,2; CI 95%: 1,1 – 4,3) in females. It should be with any of the haplotypes), because the risk for depressed noted that the "A" allele of rs1181252 only had a popula- state due to the male gender is differs in the analyses of the tion frequency of 6%. MAOA locus (OR = 1,4; non-significant) and the MAOB locus, where the estimate is similar to the model without Associations with haplotypes genetic information. There was no association between any of the MAOB hap- lotypes and trbc-MAO activity. Two MAOA haplotypes, A1 Interestingly, in females all MAOB homozygote haplo- and A3, both sharing identical alleles at the three first hap- types displayed greater odds ratios with depressed state lotype positions (CCA-) (Figure 1), were associated with a than that for heterozygotes (Table 2), indicating an addi- significant decrease in trbc-MAO activity (Table 1). Analy- tive effect. The same was true for MAOA (Table 2). Page 3 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 LD map Figure 2 LD map. Pair-wise LD map with one individual from each female pair (N = 356). D' is shown below the diagonal and ∆ above the diagonal. Color code D': Red: ≥0,8 Orange: 0,5–0,8 Yellow: 0,3–0,5 White: <0,3. Color code ∆ : Red: ≥0,30 Orange: 0,1– 0,30 Yellow: 0,05–0,1 White: <0,05. state. On the other hand, there was an interesting, Discussion Monoamine oxidase A and B constitute two important although not significant dose-response effect of haplo- molecules in the human body in general and in the types displayed in women, with greater odds ratios in central nervous system (CNS) in particular. Numerous homozygotes than heterozygotes. studies suggest a contribution of these two mitochondrial enzymes to complex human behaviors [26-28,31-33]. In Considering the size and importance of the MAO locus, the present study we searched the MAO locus for novel relatively few polymorphic sites have been verified. We genetic variants and evaluated the genetic and haplotype observed two new variants through sequence screening a structure in a Swedish population. We also assessed asso- partial region of MAOA intron 1, but in MAOB neither the ciations between trbc-MAO activity and depressed state, previously reported nor any novel variants were found in and their respective associations with the genetic structure the areas sequenced. It is surprising that so few SNPs were of the MAO locus. The key findings of this study are first: discovered given our power to detect variants with very the profound lack of variation at functional regions of the low frequencies. SNP deserts have been previously noted two MAO genes and a pattern of two distinct genetic LD on the q arm of the X chromosome [34]. Gilad and col- blocks, one for each gene. Second: we replicated the gen- leagues [4] have described similar features across MAOA, der differences in trbc-MAO activity and demonstrated an where extensive LD and low nucleotide diversity suggest association between trbc-MAO activity and depressed recent action by population structure forces and perhaps a state in women. Third: two MAOA haplotype variants recent positive selection sweep [35]. Although we could were associated with decreased trbc-MAO activity not evaluate the influence of such forces, evidence of although we could not replicate a previously reported strong LD and the lack of decay across MAOA in our Swed- genetic association between the MAOB gene and trbc- ish sample complement these previous findings. Linkage MAO activity. Fourth: we could not find any significant disequilibrium decays rapidly between the two MAO associations between the genetic variants and depressed genes (separated by approximately 20 kb). Perhaps selec- Page 4 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Table 1: Associations between MAO haplotypes and trbc-MAO activity, reported as unit change in mean trbc-MAO activity per allele and controlling for gender and smoking status. Total sample N = 340 Males N = 156 Females N = 184 per allele Hemizygous per allele Haplotypes Estimates (Unit change in mean trbc-MAO activity per allele) B1 -0,38 (-1,3 – 0,5) 0 (ref) -0,3 (-1,1 – 0,5) B2 -0,63 (-1,8 – 0,5) -0,08 (-1,6 – 1,5) -0,4 (-1,5 – 0,7) B3 -0,18 (-1,6 – 1,3) -0,8 (-2,9 – 1,2) -0,2 (-1,5 – 1,0) B4 -1,3 (-3,5 – 0,8) 0,7 (-2,8 – 4,3) -1,0 (-2,8 – 0,8) B5 -1,7 (-5,1 – 1,6) NA -0,7 (-3,8 – 2,4) Male gender -2,1 (-3,3 – -0,9)* Non-smokers 2,3 (1,1 – 3,5)* 1,5 (0,2 – 2,8) 3,5 (2,0 – 5,0) A1 -1,1 (-1,9 – -0,3)* -1,8 (-3,2 – -0,5)* -1,0 (-1,7 – -0,3)* A2 0,1 (-0,9 – 1,2) 0 (ref) 0,6 (-0,4 – 1,5) A3 -3,1 (-6,1 – -0,14)* -2,3 (-5,8 – 1,2) -4,1 (-7,4 – -0,7)* A4 -0,02 (-3,8 – 3,7) NA -0,5 (-3,7 – 2,7) Male gender -2,3 (-3,5 – -1,1)* Non-smokers 2,4 (1,1 – 3,6)* 1,4 (0,03 – 2,8) 3,4 (1,9 – 4,8) tion is in action much more locally than would be other hand, it is possible that the MAOA locus holds cis- expected in each MAO gene, both separated by regions of acting regulatory elements affecting MAOB expression. higher recombination than that within each gene. Another possible explanation could be that one or several single-base variants affected by methylation cause Previous studies have indicated that the MAOA gene may changes in the expression pattern [40]. harbour relatively few haplotypes within a block structure [5]. We observed similar results here with two haplotypes Our study is based on a relatively large population-based encompassing 95% of the haplotypic variation. We found sample of normally aging adults, although it is not with- similar results for the MAOB gene, with a distinct block out its limitations. We have controlled for smoking, but structure in which three haplotypes explain 93% of the were unable to do so for intake of certain medications. variation. So few haplotypes over such long distances The study sample was included in a larger study where have been observed previously (McCarthy et al, manu- associations between depressed state and the serotonin script) and are proposed signatures of selection and pop- receptor 2A and the serotonin transporter were evaluated ulation substructure on the X chromosome [36,37]. [41]. The influence of these genes has not been corrected for in the analysis. A previous Swedish association between the MAOB gene and trbc-MAO activity [21] could not be replicated nor Conclusion distinctly refuted, as we found a small non-significant Good et al [19] demonstrated that trbc-MAO activity is effect of the same allele in males. However, none of the related to the number of X chromosomes. We replicated a haplotype blocks carrying this allele could strengthen or significant difference in trbc-MAO activity between males support this effect, suggesting that this allele is not in high and females reported by others e.g. [17]. The findings sug- LD with a larger region of the MAOB gene. gest incomplete X chromosome inactivation at this locus and are consistent with other findings of genes escaping Two MAOA haplotypes (A1 and A3) showed a significant inactivation on the X chromosome [42,43]. It has been association with reduced trbc-MAO activity. Both haplo- hypothesized that this dosage imbalance between males types shared the initial sequence variants [CCA], but var- and females might be crucial for gender characteristics ied at the fourth allele [T/C]. Given that only MAOB is [19,44]. Recently it was demonstrated that a number of expressed in platelets there is no clear explanation for this genes, including MAOA, escape X-inactivation [45]. Fur- finding. Given the minor kinetic differences between thermore, the X-inactivation pattern, which shows a sub- platelet and brain MAO-B [38] and the correlation of stantial heritability [46], increases in the elderly. Although MAOB and MAOA levels in regions of the brain [39], this we could not find a significant association between vari- association may reflect MAOA activity in the brain. On the ants of MAOB or MAOA and depressed state in this popu- Page 5 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Table 2: MAO haplotypes and depressive state, reported as odds ratios per allele. Without genetic information in the model male gender was significant [OR: 0,5 (0,3 – 0,8)] for depressive state. *Homozygote compared to heterozygote. MAO haplotypes and depressive state Total sample N = 573 Males N = 239 Females N = 334 per allele Hemizygous per allele Homozygotes* Odds Ratio with 95% CI B1 1,2 (0,6 – 2,5) 1,0 (ref) 1,4 (0,6 – 2,9) 1,3 p = 0,57 B2 1,5 (0,7 – 3,3) 1,7 (0,8 – 3,6) 1,5 (0,6 – 3,3) 1,2 p = 0,80 B3 1,3 (0,5 – 3,0) 0,7 (0,3 – 1,7) 1,7 (0,7 – 4,2) 1,9 p = 0,51 B4 2,0 (0,8 – 5,2) 3,7 (0,7 – 18,7) 2,0 (0,7 – 5,3) 1,7 p = 0,59 B5 0,5 (0,1 – 2,8) 2,4 (0,4 – 14,5) 0,3 (0,04 – 2,5) NA Male gender 0,7 (0,3 – 1,5) A1 3,0 (0,8 – 12,2) 1,0 (ref) 2,2 (0,6 – 8,4) 5,5 p = 0,08 A2 2,5 (0,6 – 10,6) 0,9 (0,4 – 1,8) 1,7 (0,4 – 7,1) 1,3 p = 0,80 A3 2,8 (0,7 – 11,4) 1,2 (0,3 – 4,4) 1,7 (0,4 – 6,9) NA A4 3,2 (0,6 – 18,6) NA 2,8 (0,4 – 17,3) NA Male gender 1,4 (0,3 – 6,0) lation, we found an interesting dose-response effect in blood donors were randomly selected from a larger sam- women, with a higher risk for depressed state with ple set collected to study MAOB regulation. All were homozygosity. Whether levels of trbc-MAO activity are between the ages of 20 to 40 years and non-smokers. correlated with the number of X chromosomes and whether this might be linked to the higher prevalence of This study was reviewed and approved by the Ethics Com- depressive symptoms in females deserves further investi- mittee of the Karolinska Institute, the Swedish Data gation. Nevertheless it is plausible that a partially doubled Inspection Board, and the IRBs at the University of South- gene activity on the X chromosome can explain difference ern California and the Pennsylvania State University. All in prevalence of depressive state in men and women. subjects provided informed consent. DNA and trbc-MAO activity Methods Participants DNA samples were available from 573 twins. Trbc-MAO The participants were taken from a longitudinal twin activity measures were available from 565 twins. The trbc- study of aging, the Swedish Adoption/Twin Study of MAO activity is expressed as nmoles of 2-phenylethyl- platelets. Trbc- Aging (SATSA) with up to five occasions of measurement amine oxidized per minute and per 10 [47]. SATSA is a sub-sample of the population based MAO activity measures have previously been described in Swedish Twin Registry [48]. All participants are Caucasian detail [20]. and born in Sweden. For the present analyses we selected Depressed state all individuals who participated in an in-person testing session during which questionnaires were administered Depressive symptoms were measured with the Center for and a blood sample was drawn. The mean age of the sam- Epidemiologic Studies Depression Scale (CES-D), a 20- ple was 61,3 years at the time of testing. Twenty two per- item self-report instrument developed for use in the com- cent of the participants were current smokers; 35% of the munity and well established for use with older adults males and 15% of the females. [49,50]. The scale has been shown to have minimal over- lap with physical illness [51] and assesses current symp- Zygosity was initially based on self-reports of similarity toms during the past week. Respondents scoring 16 or and confirmed by serological analyses and comparisons higher on the CES-D scale are considered to have a clini- of up to 10 DNA markers. cally relevant depressed state. In this study population of 574 participants, 144 were classified as having a depressed For preliminary screening of the promoter, the first exon state, 17.9% of the males and 30.2% of females. and intron regions for novel variants, 94 Swedish male Page 6 of 9 (page number not for citation purposes) BMC Genetics 2005, 6:46 http://www.biomedcentral.com/1471-2156/6/46 Genotyping & sequencing matics.org/macroshack/programs/PHARE) to create input Approximately 4.5 kb of each gene was initially sequenced files for "PHASE" [59,60] to construct female haplotypes. in search of novel SNPs in both MAOA and MAOB, first in 94 Swedish males and later 45 twins with CES-D scores We used linear regression to estimate the association (36 males and 9 females). Power to detect minor allele fre- between trbc-MAO activity and genotypic information quencies (q) between 1 and 5% was determined as by using a generalized estimating equation (GEE) approach Glatt et al. [52], 1-(1-q) where N is the number of chro- and alternating logistic regression (ALR) [61] to estimate mosomes. Amplification and nested sequencing primers the association between depressed state and genotypic were designed with the CPrimers programme from Gen- information. We first modeled the association between bank entry GI:8671203 containing the promoter, coding single SNPs and each of the two outcomes and then mod- exon 1 and flanking intronic sequence of MAOA (~5.0 kb, eled the association between haplotype constructs and the nucleotides 46490–51454) and Genbank entry two outcomes. All estimates were adjusted for current GI:2440066 spanning the same characterized sequences smoking status. We estimated both dominance and co- of MAOB (~4 kb nucleotides 35033–39021). dominance models. Explanatory variables in the domi- nance models were binary whereas in the co-dominance Direct sequencing reactions were performed using models they were coded as the number of reference alleles DYEnamic ET Dye Terminator Cycle Sequencing Kit (i.e., 0, 1, or 2 for females and 0 or 1 for males). The (Amersham Biosciences) and separated using a Megabace parameter estimates for the co-dominance models repre- 1000. Reads were base called with Phred [53], assembled sent the change in the outcome (trbc-MAO activity or using Phrap and viewed using Consed Version 13 [54]. All odds of being in a depressed state) per affected allele. Due SNPs were documented and cross validated with dbSNP at to the continuous nature of the trbc-MAO measure, only NCBI. one individual from each complete twin pair and single participating individuals were analyzed (N = 340). Twelve SNPs identified from public databases (dbSNP at Among females we also estimated the effect of being NCBI) and two novel SNPs previously reported (introns 3 homozygote compared to heterozygote. If the co-domi- and 10 of MAOB) a Swedish sample [5] were sequenced nance model is a good fit to the data then these estimates in 95 participants (142 chromosomes) by Pyrosequencing should be similar to the "per allele" estimates from the co- to confirm their presence in this population. For Pyrose- dominance model. All statistical analyses were performed quencing, either the forward or the reverse primer in each in SAS 8.01 using GENMOD procedure (SAS Institute Inc. primer pair was biotinylated. Sequencing primers with a Cary, NC). length of 14 and 18 bases were placed within one base of the SNP. The PCR reaction was performed in a 50 µl reac- Authors' contributions tion volume, containing 5 ng of genomic DNA, 10 pmoles MJ: Design of the study, performed data analysis and of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl and interpretation of data. Carried out the molecular genetic 1.5 U of Taq. Thermal cycling was performed in a PTC-225 studies (genotyping) and drafted the manuscript. DNA machine (MJ Research Inc., Cambridge, MA, USA) at 95°C for 5 min followed by 50 cycles of 95°C for 30 s, 45 SM: Participated in the design of the study. Carried out the s of annealing at an optimized temperature, followed by molecular genetic studies (sequencing), sequence align- 72°C for 30 s and a final extension of 5 min at 72°C. The ment and critically revised the manuscript. biotinylated PCR product was immobilized onto strepta- vidin-coated sepharose beads and DNA strands were sep- PFS: Participated in the design of the study and critically arated by denaturation with 0.2 M NaOH. The revised the manuscript for important intellectual content. pyrosequencing reaction was performed on a PSQ96™ Instrument from Pyrosequencing AB (Uppsala, Sweden) PD: Planed and performed the statistical analysis. as described by [55,56]. Detailed primer and assay infor- mation are available upon request. BA: Participated in the design of the study and critically revised the manuscript. Statistical analysis Male haplotypes could be extrapolated directly since the LO: Substantially revised the manuscript for important MAO locus is located on the X chromosome and males are intellectual content. thereby hemizygous. Female bi-allelic haplotypes were estimated using an EM algorithm (Sham 1998) and the MS: Participated in the design of the study and critically pair-wise LD measures D' [57] and ∆ [58]. We used revised the manuscript. 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